Antibody to human il-3 receptor alpha chain

ABSTRACT

The present invention provides an antibody to human IL-3Rα chain, which does not inhibit IL-3 signaling and binds to B domain of the human IL-3Rα chain but does not bind to C domain of the human IL-3Rα chain; a composition for preventing or treating a blood tumor in which a cell expressing IL-3Rα is found in bone marrow or peripheral blood of a subject, which comprises the antibody to human IL-3Rα as an active ingredient; and a method for treating a blood tumor in which a cell expressing IL-3Rα is found in bone marrow or peripheral blood, which comprises administering, to a subject, a composition comprising the IL-3Rα antibody as an active ingredient.

TECHNICAL FIELD

This invention relates to an antibody to human IL-3Rα protein (anothername: human CD123). The invention also relates to an invention of atherapeutic agent and diagnostic agent for myelocytic malignant tumors,particularly acute myeloid leukemia (AML), which comprises a humanIL-3Rα antibody as an active ingredient.

BACKGROUND OF THE INVENTION Regarding Malignant Tumor:

A malignant tumor (cancer) is the first leading cause of death in Japanand the number of patients is increasing every year, and the developmentof a drug and a therapeutic method having high efficacy and safety isstrongly desired. As the cause of forming a malignant tumor, there is amutation of DNA by radiation, ultraviolet rays and various carcinogenicsubstances. Studies on malignant tumors have been focused on molecularbiological identification of these genetic changes. As a result, it isconsidered that tumorigenic transformation is induced by accumulation ofa large number of mutations and the like. It has been shown by a cellline model and the like that some decisive mutations directly connectedwith the tumorigenic transformation. Regarding leukemia as one of theobjective diseases of the invention, many chromosomal abnormalities havebeen identified and classified. In many of the case, translocation ofchromosome is found and the some genes associated with chromosomaltranslocation have already been identified in principle chromosomaltranslocations. By analyses of functions of the translocation relatedgenes, a case has been found that these genes are concerned in the onsetof leukemia.

Regarding Cancer Stem Cell:

On the other hand, a so-called cancer stem cell hypothesis has beenproposed for a long time from the viewpoint of cell biology, statingthat stem cell is the origin of a malignant tumor similar to the normaltissue. The stem cell is defined as a cell having autonomous replicationability and pluripotency and generally divided roughly into totipotencystem cell and tissue stem cell. Tissue stem cells are originated fromspecific tissues and organs such as of blood system, liver, nerve systemand the like and present at an extremely low frequency. Among them,hematopoietic stem cell has been studied most frequently. It has beenreported that a hematopoietic system can be reconstructed over a longperiod of time by transplanting one hematopoietic stem cell into a mousein which the hematopoietic system was destructed by a lethal dose ofirradiation (Non-patent Document 1). Different from the normal stemcell, studies on cancer stem cells have been delayed for a prolongedperiod of time since their true nature could not been found. However, acancer stem cell has been identified for the first time in acute myeloidleukemia, in 1997 by Dick et al. Thereafter, the presence of cancer stemcells has been reported in various malignant tumors. In summing up,cancer stem cells are present at a frequency of several % or less of thewhole tumor and rare as well as normal stem cells. It is considered thatthe remaining cells which form the tumor are tumor precursor cells inwhich proliferation ability is limited or tumor cells.

By these reports, it was shown that hierarchy is present even in tumorsimilar to the normal tissue, and the cancer stem cell residing at thispeak (origin) has strong tumor forming ability.

Characteristics and Therapeutic Problems of Cancer Stem Cells:

In summing up many reports, it is considered that cancer stem cells aremaintaining various characteristics possessed by the normal stem cells.Examples of similarities include the rarity of the cells, amicroenvironment (niche) in which the cell exists, expression of amultiple drug resistance gene, cell cycle arrest, and the like.

Particularly, the characteristics that they express a group of multipledrug resistance genes and are at the interphase of cell cycle similar tothe normal stem cells could become a therapeutically great problem. Amultiple drug resistance gene BCRP is a pump which impairs the drugefficacy by eliminating various antitumor agents into outside of cells,and a method for collecting stem cells making use of the activity hasbeen reported (Non-patent Document 2). In addition, their presence atthe interphase of cell cycle under a state of “arresting” (Non-patentDocument 3) is causing reduction of sensitivity for many antitumoragents and radiations which targets the quick cell growth of cancer(Non-patent Documents 4 and 5).

Based on the above characteristics, it is considered that the cancerstem cell which exhibiting resistance to the therapy is a cause of tumorregeneration.

Regarding Molecular Target Drug

Three main courses of the treatment of a malignant tumor include ofantitumor agent therapy, radiation therapy and surgical excision. Theblood tumor is limited to the antitumor agent therapy and radiationtherapy, and as described in the above, the cancer stem cell can have aresistance to these treatments. Another problem is that side effects arelarge since these two treatments affect the entire body. It is amolecular target drug that is expected as a resolving means for thisproblem. It has a possibility to reduce side effects by exhibiting itsdrug efficacy only in the cell expressing the target molecule.

Examples of typical drugs of the molecular target drug in the field ofblood diseases include imatinib and rituximab. Imatinib targets at aleukemia-causing factor called Bcr-Abl produced by a chromosomalabnormality (Philadelphia chromosome) which is observed in 95% of CMLpatients. This is a low molecular weight drug which induces suicide ofleukemia cell by inhibiting function of Bcr-Abl. Rituximab is atherapeutic antibody which recognizes CD20 as a surface molecule on a Bcell and has an antitumor effect on a malignant tumor of B cell(non-Hodgkin lymphoma and the like). On the other hand, molecular targetdrugs for AML are few, and there is only an agent gemtuzumab.ozogamicin(Mylotarg) in which an antibiotic calicheamicin is linked to amonoclonal antibody for CD33 known as an AML cell surface antigen.However, it is the present situation that the use of Mylotarg is limitedbecause of its strong toxicity which is considered to be derived fromcalicheamicin in addition to the problem that therapeutic range isnarrow. Based on the above, it can be said that discovery of a newtarget gene and development of a therapeutic agent for this areimportant inventions which directly lead to the possibility of therapyand expansion of the choices of therapy.

As the embodiment of molecular target drugs, various substances havebeen studied and developed such as a therapeutic antibody and a lowmolecular weight drug, as well as a peptide drug, a biological proteinpreparation such as cytokine, an siRNA, aptamer and the like. When anantibody is used as a therapeutic agent, due to its specificity, it isuseful in treating pathological conditions in which the disordered cellexpresses a specific antigen. The antibody binds to a protein expressingon the cell surface as its antigen and effectively acts upon the boundcell. The antibody has a characteristic of long blood half life and highspecificity for its antigen and is also markedly useful as an antitumoragent. For example, when an antibody targets at a tumor-specificantigen, it can be expected that the administered antibody accumulatesinto the tumor and thereby attacks the tumor cell viacomplement-dependent cytotoxicity (CDC) and antibody-dependent cellularcytotoxicity (ADCC). In addition, by binding a radioactive substance, acytotoxic substance and the like to an antibody, it becomes possible totransfer an agent efficiently to the tumor part and thereby to allow toact thereon. At the same time, it can decrease the amount of the reachedagent to non-specific other tissues and reduction of side effects canalso be expected. Inhibition of tumor growth or regression of tumor canbe expected by administering an antibody having agonistic activity whena tumor-specific antigen has an activity to induce cell death, or byadministering an antibody having neutralization activity when atumor-specific antigen relates to in the growth and survival of cells.Due to the above characteristics, it is considered that antibodies aresuited in applying as antitumor agents.

Regarding Therapeutic Antibodies:

In the original antibody preparation, a mouse was used as the animal tobe immunized. However, use of mouse antibodies as drugs is limited dueto a large number of reasons. A mouse antibody which can be recognizedas a foreign substances in the human body can induce so-called “humananti-mouse antibody” namely “HAMA” response (Non-patent Document 6).Further, the Fc region of mouse antibody is not effective for the attackon disease cells via human complement or human immune cells.

As one of the approaches for avoiding such problems, a chimeric antibodyhas been developed (Patent Documents 1 and 2). The chimeric antibodycontains parts of antibodies derived from two or more species (mouseantibody variable region, human antibody constant region and the like).An advantageous point of such a chimeric antibody is that it keeps thecharacteristics of mouse antibody but can stimulate human complement orhuman immune cells since it has human Fc. However, it is known that sucha chimeric antibody still induces “human anti-chimeric antibody” namely“HACA” response (Non-patent Document 7).

Further, it has been developed a recombinant antibody in which only acomplementarity determining regions (“CDRs”) of a part of an antibodywere substituted (Patent Documents 3 and 4). By the use of a CDRgrafting technique, an antibody comprising mouse CDR and human variableregion framework and human constant region, so-called “humanizedantibody” (Non-patent Document 8). Further, by the use of a humanantibody producing mouse or by a screening using a human antibodylibrary, broadly utilized techniques have been provided also regardingpreparation of complete human antibodies (Non-patent Documents 9 and10).

Regarding IL-3Rα:

IL-3Rα is the α chain of IL-3 receptor, belongs to a cytokine receptorfamily and shows weak affinity for IL-3 as its ligand. By forming ahetero receptor with its β chain (CD131, hereinafter also referred to asIL-3Rβ), an IL-3 receptor has a strong binding and transfers a signalsuch as growth, differentiation and the like into a cell throughintracellular region of the β chain. IL-5 receptor α chain and GM-CSFreceptor α chain share the β chain in common.

IL-3Rα is a type I membrane protein of single-pass transmembrane, and itis known based on the sequence that an IL-3 binding site and afibronectin type III site are present in the extramembrane region. It isknown that there is no structure which can transfer a signal in theintramembrane region. Though three-dimensional structure of IL-3Rα hasnot been analyzed yet, it can be assumed that structures of cytokinereceptors are similar between families since position of cysteineresidue which forms the structurally important S—S bond is preserved inmost cases. Among the same cytokine receptors, crystalline structures ofIL-13 receptor α chain, IL-4 receptor α chain and GM-CSF receptor αchain have been analyzed. Based on the information of these cytokinereceptor families, it can be assumed that the extramembrane region ofIL-3Rα is roughly divided into 3 domains (A-B-C domains). It is knownthat an antibody 7G3 which recognizes A domain of human IL-3Rα blocksIL-3 signaling (Non-patent Document 11). In addition, expression of an Adomain-deficient IL-3Rα molecule has been reported (Non-patent Document12), and as a matter of course, an antibody which recognizes A domaincannot recognize A domain-deficient IL-3Rα. In addition, it isconsidered that C domain is the root of IL-3Rα molecule and has a highpossibility to three-dimensionally inhibit association of IL-3Rβ withIL-3Rα.

IL-3 is the only a ligand which is known as a ligand of IL-3Rα. IL-3 isa hematopoietic factor which is known to accelerate colony formation ofthe following: erythrocyte, megakaryocyte, neutrophil, eosinophil,basophil, mast cell and a monocyte system cell. It is known that IL-3also stimulates a precursor cell having pluripotency, but IL-3 is rathersaid to accelerate a differentiation of not an immature stem cell havingautonomous replication ability but a precursor cell committed todifferentiation.

It is known that IL-3Rα relates to the growth and differentiation ofmyeloid cells by forming a heterodimer with β chain and therebytransferring the IL-3 signaling into the cell via the Serine/Threoninephosphorylation pathway. It is known that IL-3Rα is expressed inGranulocyte-Macrophage Progenitor (GMP) or Common Myeloid Progenitor(CMP) among hematopoietic precursor cells and induces growth anddifferentiation into neutrophil and macrophage systems via the IL-3signaling. On the other hand, it has been reported that theMegakaryocyte Erythroid Progenitor (MEP) presenting in the downstream ofCMP does not express IL-3Rα different from the GMP which is also presentin the downstream.

Regarding the AML stem cell, Bonnet and Dick have reported that the AMLstem cell is present in the CD34 positive CD38 negative fraction(Non-patent reference 13). Further, by comparing with the same fraction(CD34 positive CD38 negative) of normal stem cell, Jordan et al. havefound that IL-3Rα is highly expressed in the AML stem cell (Non-patentreference 14). A high potential of IL-3Rα as a marker of not only AMLstem cell but also leukemia stem cell has also been reported in theplural of reports thereafter (Non-patent references 15 and 16). In thetreatment of cancers including leukemia, it is important that only thecancer cells are removed without injuring normal cells as many aspossible, and it is considered that this difference in the expression ofIL-3Rα between normal stem cell and leukemia stem cell is useful in thetreatment targeting at the leukemia stem cell.

Regarding IL-3Rβ which forms a heterodimer with IL-3Rα, there is noreport that IL-3Rβ is highly expressed leukemia stem cell, and also inthe case of a microarray in which expression of mRNA in leukemia stemcell and normal stem cell is compared in fact, IL-3Rβ is not identifiedas a molecule in which its expression is increased in leukemia stem cell(Non-patent reference 17).

Regarding IL-3Rβ which forms a heterodimer with IL-3Rα, there is noreport that IL-3Rβ is highly expressed leukemia stem cell, and also inthe case of a microarray in which expression of mRNA in leukemia stemcell and normal stem cell is compared in fact, IL-3Rβ is not identifiedas a molecule of which expression is increased in leukemia stem cell(Non-patent reference 18).

The presence of a leukemia cell which depends on IL-3 has been known fora long time, and the old studies are studies focused on a blast cellwhich occupies most of the leukemia cells. According to the recentstudies on leukemia stem cell, it is said that the leukemia stem cellacquires antitumor agent resistance by exhaustively suppressing itsgrowth. In addition, it is considered that an IL-3 reactive blast cellhas high proliferation ability so that it is assumed that such a cell iseffective in the general treatment using an antitumor agent.

As a candidate of the agent targeting at an IL-3R receptor, the IL-3itself was administered for a long time to patients of hematopoieticinsufficiency but it did not become a drug as a result. A clinical trialfor a fusion protein in which diphtheria toxin is added to IL-3 is inprogress aiming leukemia as a target of the disease. Regarding the IL-3and diphtheria toxin-IL-3 fusion, these are not suitable as the agentswhich are targeting at cells in which expression of IL-3Rα isspecifically increased, since IL-3 binds strongly not a protein ofIL-3Rα alone but a hetero protein of IL-3Rα and β due to properties ofIL-3. On the other hand, as a candidate of an agent targeting at IL-3Rα,a first phase result of an IL-3Rα human mouse chimeric antibody 7G3 hasbeen reported (Non-patent Document 19). Since the 7G3 chimeric antibodyuses for the purpose of blocking of IL-3 signaling as the mechanism ofAML therapy, this is not an agent aimed at removing IL-3Rα positivecells. Also, although some other IL-3Rα antibodies are known (9F5(Becton Dickinson), 6H6 (SANTA CRUZ BIOTECHNOLOGY) and AC145(Miltenyi-Biotech)), these do not have the ability to remove the cellshighly expressing IL-3Rα.

CITATION LIST Patent Document

-   Patent Document 1: EP Published Patent Application 120694-   Patent Document 2: EP Published Patent Application No. 125023-   Patent Document 3: GB Patent application No. GB2188638A-   Patent Document 4: U.S. Pat. No. 5,585,089

Non-Patent Document

-   Non-patent Document 1: Osawa M et al., Science. 273:2 42-5 (1996)-   Non-patent Document 2: Goodell M A et al., J Exp Med. 183: 1797-806    (1996)-   Non-patent Document 3: Yamazaki S et al., EMBO J. 25: 3515-23 (2006)-   Non-patent Document 4: Ishikawa F et al., Nat. Biotechnol.    25:1315-21. (2007)-   Non-patent Document 5: Bao S et al., Nature. 444: 756-60 (2006)-   Non-patent Document 6: Schiff et al., Canc. Res., 45, 879-885 (1985)-   Non-patent Document 7: Bruggemann et al., J. Exp. Med.,    170:2153-2157 (1989)-   Non-patent Document 8: Riechmann et al., Nature, 332:323-327 (1988)-   Non-patent Document 9: Ishida I et al., Cloning Stem Cells. 4:91-102    (2002)-   Non-patent Document 10: Wu et al., J Mol Biol. 19:151-62 (1999)-   Non-patent Document 11: Sun et al., Blood, 87:83 (1996)-   Non-patent Document 12: Chen et al., J Biol Chem, 284: 5763 (2009)-   Non-patent Document 13: Bonnet et al., Nat Med, 1997; 3: 730-   Non-patent Document 14: Jordan et al., Leukemia, 2000; 14: 1777-   Non-patent Document 15: Haematologica, 2001; 86:1261-   Non-patent Document 16: LeukLymphoma, 2006; 47:207-   Non-patent Document 17: Majeti et al., Proc Natl Acad Sci USA. 2009;    106:3396-   Non-patent Document 18: Majeti et al., Proc Natl Acad Sci USA.    106:3396 (2009)-   Non-patent Document 19: Blood, 2008 112 (11): Abstract 2956

SUMMARY OF THE INVENTION Technical Problems

An object of the invention is to provide a therapeutic agent which canremove leukemia stem cells alone and also can hardly exhibit adverseeffects upon normal cells (shows fewer side effects). Specifically, thepresent invention provides an antibody to human IL-3Rα chain, which doesnot inhibit IL-3 signaling and binds to B domain of human IL-3Rα chainbut does not bind to C domain; a composition comprising the antibody;and a therapeutic method or detection method comprising the antibody.

Solution to Problems

The invention relates to the following (1) to (9).

(1) An antibody to a human IL-3Rα chain, which does not inhibit IL-3signaling and binds to B domain of human IL-3Rα chain but does not bindto C domain.

(2) The antibody described in the above-mentioned (1), further havinghigh antibody-dependent cellular cytotoxicity (ADCC).

(3) The antibody described in the above-mentioned (1) or (2), whereinthe high antibody-dependent cellular cytotoxicity (ADCC) shows aspecific lysis rate of 10% at an antibody concentration of 0.01 μg/ml,by a Colon-26/hCD123 ADCC assay method which uses PBMC cultured withIL-2.

(4) The antibody described in any one of the above-mentioned (1) to (3),which comprises amino acid sequences of CDRs of heavy chain and CDRs oflight chain selected from the group consisting of the following (a) to(e);

(a) CDR 1 to 3 of heavy chain are the amino acid sequences of SEQ IDNOs:113 to 115, respectively, and CDR 1 to 3 of light chain are theamino acid sequences represented by SEQ ID NOs:131 to 133, respectively,(b) CDR 1 to 3 of heavy chain are the amino acid sequences of SEQ IDNOs:116 to 118, respectively, and CDR 1 to 3 of light chain are theamino acid sequences represented by SEQ ID NOs:134 to 136, respectively,(c) CDR 1 to 3 of heavy chain are the amino acid sequences of SEQ IDNOs:119 to 121, respectively, and CDR 1 to 3 of light chain are theamino acid sequences represented by SEQ ID NOs:137 to 139, respectively,(d) CDR 1 to 3 of heavy chain are the amino acid sequences representedby SEQ ID NOs:122 to 124, respectively, and CDR 1 to 3 of light chainare the amino acid sequences represented by SEQ ID NOs:140 to 142,respectively, and(e) CDR 1 to 3 of heavy chain are the amino acid sequences representedby SEQ ID NOs:125 to 127, respectively, and CDR 1 to 3 of light chainare the amino acid sequences represented by SEQ ID NOs:143 to 145,respectively.

(5) The antibody described in any one of the above-mentioned (1) to (4),which comprises the heavy chain variable region and light chain variableregion selected from the group consisting of the following (a) to (f);

(a) a heavy chain variable region comprising an amino acid sequence fromglutamine (Q) at position 20 to serine (S) at position 139 in the aminoacid sequence of SEQ ID NO:53 and a light chain variable regioncomprising an amino acid sequence from valine (V) at position 23 tolysine (K) at position 129 in the amino acid sequence of SEQ ID NO:55;(b) a heavy chain variable region comprising an amino acid sequence fromglutamine (Q) at position 20 to serine (S) at position 139 in the aminoacid sequence represented by SEQ ID NO:57 and a light chain variableregion comprising an amino acid sequence from valine (V) at position 23to lysine (K) at position 129 in the amino acid sequence of SEQ IDNO:59;(c) a heavy chain variable region comprising an amino acid sequence fromglutamine (Q) at position 20 to serine (S) at position 139 in the aminoacid sequence represented by SEQ ID NO:61 and a light chain variableregion comprising an amino acid sequence from aspartic acid (D) atposition 23 to lysine (K) at position 129 in the amino acid sequence ofSEQ ID NO:63;(d) a heavy chain variable region comprising an amino acid sequence fromglutamine (Q) at position 20 to serine (S) at position 139 in the aminoacid sequence of SEQ ID NO:65 and a light chain variable regioncomprising an amino acid sequence from aspartic acid (D) at position 23to lysine (K) at position 129 in the amino acid sequence of SEQ IDNO:67;(e) a heavy chain variable region comprising an amino acid sequence fromglutamine (Q) at position 20 to serine (S) at position 138 in the aminoacid sequence represented by SEQ ID NO:69 and a light chain variableregion comprising an amino acid sequence from aspartic acid (D) atposition 23 to lysine (K) at position 129 in the amino acid sequence ofSEQ ID NO:71 and;(f) a heavy chain variable region and/or light chain variable region,which comprise amino acid sequences in which 1 to 3 amino acid residuesare deleted, substituted, added or inserted in the heavy chain variableregion and/or light chain variable region shown by the above (a) to (e).

(6) A composition for preventing or treating a blood tumor in which acell expressing IL-3Rα is found in bone marrow or peripheral blood of asubject, which comprises the IL-3Rα antibody described in any one of (1)to (5) as an active ingredient.

(7) A method for treating a blood tumor in which a cell expressingIL-3Rα is found in bone marrow or peripheral blood, which comprisesadministering, to a subject, a composition comprising the IL-3Rαantibody described in any one of (1) to (5) as an active ingredient.

(8) A composition for detecting a blood tumor in which a cell expressingIL-3Rα is found in bone marrow or peripheral blood of a biologicalsample from a subject, which comprises the IL-3Rα antibody described inany one of (1) to (5).

(9) The composition or method described in any one of (1) to (5),wherein the aforementioned blood tumor is acute myeloid leukemia (AML).

Advantageous Effect of the Invention

The invention can provide an antibody to human IL-3Rα chain, which doesnot inhibit IL-3 signaling and binds to B domain of human IL-3Rα chainbut does not bind to C domain; a composition which comprises saidantibody and a therapeutic method or detection method using saidantibody.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 and 2 are results of a flow cytometry analysis of a cellexpressing an IL-3Rα/GM-CSFRα chimeric protein using a labeledanti-IL-3Rα antibody.

FIG. 3 is a result of a flow cytometry analysis of a cell expressing anIL-3Rα/GM-CSFRα chimeric protein using a labeled anti-IL-3Rα antibody.

FIG. 4 is a graph in which, among the nucleotide and amino acidsequences of A and B domains of human IL-3Rα molecule, parts of regionsin which the regions 1 to 7 arranged on the outside of the molecule aresubstituted by the GM-CSFRα sequence are shown by dotted lines.

FIG. 5 is a result of a cell growth test for examining blocking activityof IL-3 signaling. The ordinate represents the cell growth inhibitionratio (%) and the abscissa represents various IL-3Rα antibody names.

FIG. 6 is a result of a colony assay test for examining blockingactivity of IL-3 signaling. GM, E, and GEMM show results usingGranulocyte/Macrophage system, Erythroid system colony and mixedcolonies, respectively.

FIG. 7 is a result of examining anti-tumor effects of various humanantibodies in a tumor bearing model. The ordinate represents the numberof MOLM13 cells, and the abscissa represents the various IL-3Rα antibodynames.

FIGS. 8 and 9 are results of ADCC test for IL-3Rα expressing cell linesusing anti-IL-3Rα antibody. PBMC not cultured with IL-2 was used in FIG.8, and PBMC cultured with IL-2 was used in FIG. 9.

FIG. 10 is a result of flow cytometry analysis of cells expressing aMacaca fascicularis IL-3Rα by anti-IL-3Rα antibody and a PE-labeledanti-human IgG secondary antibody. The upper column shows the cellsexpressing Macaca fascicularis IL-3Rα, and the lower column shows thecells expressing human IL-3Rα.

MODE FOR CARRYING OUT THE INVENTION Detailed Description of SpecifiedDesirable Embodiments

Headings of the sections to be used in this specification are only forthe purpose of organization and should not be interpreted as limitationto the main subject to be described. All of the cited references citedin this application are clearly incorporated by references into thisspecification for optional purposes.

(Outline)

This invention relates to an antibody to human IL-3Rα chain, which doesnot inhibit IL-3 signaling and binds to B domain of human IL-3Rα chain(hereinafter referred to as IL-3Rα) but does not bind to C domain.

IL-3 receptors (hereinafter referred to as IL-3R), particularly IL-3Rα,are expressed on the cell surface of a leukemia stem cell. In general,IL-3 receptor β chain (hereinafter referred to as IL-3Rβ) transfers IL-3signaling into the cell and therefore induces growth anddifferentiation.

Accordingly, there is a possibility that inhibition of IL-3 signalingcause side effects such as inhibition of normal hematopoietic action bya normal stem cell. Thus, as a new therapeutic method which targets atleukemia stem cell, it is preferable that the method targets at IL-3Rαand also does not inhibit IL-3 signaling.

(IL-3Rα)

A protein encoded by IL-3Rα gene is a type I transmembrane protein whichbelongs to a cytokine receptor family. In normal cells, the IL-3Rαmolecule is expressed on a part of hematopoietic precursor cells,basophil, a part of dendritic cells and the like. In the case of tumors,it is known to be expressed in a hematopoietic system cancer andleukemia. As examples of tumors which express IL-3Rα, it is known thatIL-3Rα is expressed on the blast cell of AML and CML in blastic crisisphase, and in the case of a differentiation marker-negative CD34positive CD38 negative fraction considered to be a leukemia stem cell,in AML, CML, MDS, ALL and SM. In blood, IL-3 which is a known ligand ofIL-3Rα is expressed on an activated T cell, a natural killer cell, amast cell and a part of cells of megakaryocyte system. In addition, theIL-3Rα is also called CD123. The IL-3Rα includes a mammal (e.g., theprimates and human) type IL-3Rα. The IL-3Rα sequence includespolymorphic variants. Specific examples of the full length human IL-3Rαinclude the following amino acid sequences.

(SEQ ID NO: 1) MVLLWLTLLLIALPCLLQTKEDPNPPITNLRMKAKAQQLTWDLNRNVTDIECVKDADYSMPAVNNSYCQFGAISLCEVTNYTVRVANPPFSTWILFPENSGKPWAGAENLTCWIHDVDFLSCSWAVGPGAPADVQYDLYLNVANRRQQYECLHYKTDAQGTRIGCRFDDISRLSSGSQSSHILVRGRSAAFGIPCTDKFVVFSQIEILTPPNMTAKCNKTHSFMHWKMRSHFNRKFRYELQIQKRMQPVITEQVRDRTSFQLLNPGTYTVQIRARERVYEFLSAWSTPQRFECDQEEGANTRAWRTSLLIALGTLLALVCVFVICRRYLVMQRLFPRIPHMKDPIGDSFQNDKLVVWEAGKAGLEECLVTEVQVVQKT

Specific examples of amino acid sequence of the extracellular region ofhuman IL-3Rα include the following amino acid sequence.

(SEQ ID NO: 2) MVLLWLTLLLIALPCLLQTKEDPNPPITNLRMKAKAQQLTWDLNRNVTDIECVKDADYSMPAVNNSYCQFGAISLCEVTNYTVRVANPPFSTWILFPENSGKPWAGAENLTCWIHDVDFLSCSWAVGPGAPADVQYDLYLNVANRRQQYECLHYKTDAQGTRIGCRFDDISRLSSGSQSSHILVRGRSAAFGIPCTDKFVVFSQIEILTPPNMTAKCNKTHSFMHWKMRSHFNRKFRYELQIQKRMQPVITEQVRDRTSFQLLNPGTYTVQIRARERVYEFLSAWSTPQRFECDQEEGAN TRAWRTSL

In addition, the extracellular region of IL-3Rα is divided into threedomains of A to C.

A domain comprises a region from glutamine (Q) at position 18 to serine(S) at position 100 in the amino acids of SEQ ID NO:2, and B domaincomprises from glycine (G) at position 101 to serine (S) at position 203in the amino acids of SEQ ID NO:2 and C domain that from glutamine (Q)at position 204 to leucine (L) at position 308 in the amino acids of SEQID NO:2.

Further, in A domain and B domain, the following 7 regions are arrangedon the outside of the molecule.

The region 1 is from aspartic acid (D) at position 55 to proline (P) atposition 61 in the amino acids of SEQ ID NO:2, the region 2 is fromvaline (V) at position 63 to phenylalanine (F) at position 70 in theamino acids of SEQ ID NO:2, the region 3 is from serine (S) at position91 to glutamic acid (E) at position 98 in the amino acids of SEQ IDNO:2, the region 4 is from proline (P) at position 97 to tryptophan (W)at position 104 in the amino acids of SEQ ID NO:2, the region 5 is fromcysteine (C) at position 122 to proline (P) at position 128 in the aminoacids of SEQ ID NO:2, the region 6 is from isoleucine (I) at position182 to serine (S) at position 188 in the amino acids of SEQ ID NO:2 andthe region 7 is from glycine (G) at position 192 to lysine (K) atposition 198 in the amino acids of SEQ ID NO:2.

Accordingly, examples of the antibody of the invention include anantibody which binds to an amino acid sequence of positions 101 to 203in the amino acids of SEQ ID NO:2 which is the extracellular region ofIL-3Rα, but does not bind to an amino acid sequence of positions 204 to308, and an antibody which further binds to amino acid sequences ofpositions 182 to 188 and positions 192 to 198 in the amino acid sequenceof SEQ ID NO:2.

The antibody of the invention binds to the above-mentioned specificregions of the extracellular region of IL-3Rα and does not inhibit IL-3signaling.

The term “does not inhibit IL-3 signaling” as used in the inventionmeans that it does not inhibit the intracellular signal through IL-3R byIL-3, and it includes a case in which the association of IL-3 with IL-3Ris not inhibited and the binding of IL-3Rα chain and β chain is notinhibited. Specifically, it means that the cell growth inhibition ratioshown by FIG. 5 according to the analysis in Example 8 is 40% or more,preferably 60% or more, further preferably 80% or more, when theantibody concentration is set to 10 μg/ml. According to thisspecification, the terms “blocking of IL-3 signaling” and “inhibition ofIL-3 signaling” are used as the same meaning and not discriminated, andthe blocking activity of IL-3 signaling means the ability to inhibitIL-3 signaling.

Also, the antibody of the invention has high antibody-dependent cellularcytotoxicity (ADCC) in addition to the above-mentioned properties.

The IL-3Rα antibody having ADCC activity means an antibody which bindsto a cell expressing IL-3Rα to kill the IL-3Rα-expressing cell via aneffector cell having cytotoxicity such as NK cell and the like.

The high ADCC activity means that the specific lysis rate is 10% or moreat an antibody concentration of 0.01 μg/ml or less when measured by aColon-26/hCD123 ADCC assay method which uses PBMC cultured with IL-2.

The specific lysis rate means a value obtained by measuring the lysisrate of a target cell by an antibody and specifically it can becalculated in accordance with the following Example 11.

Examples of the cell expressing IL-3Rα include blood cancer cells (acutemyeloid leukemia (AML) cells, chronic myeloid leukemia (CML) cells,myelody splastic syndromes (MDS) cells, acute lymphoid leukemia (ALL)cells, chronic lymphoid leukemia (CLL) cells, multiple myeloma (multiplemyeloma: MM) cells, systemic mastocytoma (SM) cells etc.), regulatory Tcells (such as CD4-positive CD25-positive cell), antigen presentingcells (such as dendritic cells, monocytes, macrophages and similar cellsthereto (hepatic stellate cells, osteoclasts, microglial cells, themajor epidermal phagocytic cells, dust cells (alveolar macrophages),etc.)), basophils and the like.

In addition, AML cell, CML cell, ALL cell, CLL cell, MDS cell, SM cell,MM cell, various lymphoma cells include their cancer stem cells.

The cancer stem cell is one of the cell groups constituting tumor. Forexample, in acute myeloid leukemia (AML) it is represented byLineage(−)CD34(+)CD38(−) myeloid cell. Accordingly, since the antibodyof the invention has high ADCC activity, it induces reduction orelimination of cells expressing IL-3Rα.

Also, the IL-3Rα antibody of the invention includes an IL-3Rα antibodywhich has CDRs of heavy chain and CDRs of light chain selected from thegroup consisting of the following (a) to (e);

(a) CDR 1 to 3 of heavy chain are the amino acid sequences representedby SEQ ID NOs:113 to 115, respectively, and CDR 1 to 3 of light chainare the amino acid sequences represented by SEQ ID NOs:131 to 133,respectively,(b) CDR 1 to 3 of heavy chain are the amino acid sequences representedby SEQ ID NOs:116 to 118, respectively, and CDR 1 to 3 of light chainare the amino acid sequences represented by SEQ ID NOs:134 to 136,respectively,(c) CDR 1 to 3 of heavy chain are the amino acid sequences representedby SEQ ID NOs:119 to 121, respectively, and CDR 1 to 3 of light chainare the amino acid sequences represented by SEQ ID NOs:137 to 139,respectively,(d) CDR 1 to 3 of heavy chain are the amino acid sequences representedby SEQ ID NOs:122 to 124, respectively, and CDR 1 to 3 of light chainare the amino acid sequences represented by SEQ ID NOs:140 to 142,respectively, and(e) CDR 1 to 3 of heavy chain are the amino acid sequences representedby SEQ ID NOs:125 to 127, respectively, and CDR 1 to 3 of light chainare the amino acid sequences represented by SEQ ID NOs:143 to 145,respectively.

In addition, the antibody of the invention includes an IL-3Rα antibodywhich comprises the heavy chain variable region and the light chainvariable region selected from the group consisting of the following (a)to (f) (shown in parentheses are names of the antibodies which aredescribed in the following Examples from which each of variable regionsare derived);

(a) a heavy chain variable region comprising an amino acid sequence fromglutamine (Q) at position 20 to serine (S) at position 139 in the aminoacid sequence represented by SEQ ID NO:53 and a light chain variableregion comprising an amino acid sequence from valine (V) at position 23to lysine (K) at position 129 in the amino acid sequence represented bySEQ ID NO:55 (name of antibody: Old4)(b) a heavy chain variable region comprising an amino acid sequence fromglutamine (Q) at position 20 to serine (S) at position 139 in the aminoacid sequence represented by SEQ ID NO:57 and a light chain variableregion comprising an amino acid sequence from valine (V) at position 23to lysine (K) at position 129 in the amino acid sequence represented bySEQ ID NO:59 (name of antibody: Old5)(c) a heavy chain variable region comprising an amino acid sequence fromglutamine (Q) at position 20 to serine (S) at position 139 in the aminoacid sequence represented by SEQ ID NO:61 and a light chain variableregion comprising an amino acid sequence from aspartic acid (D) atposition 23 to lysine (K) at position 129 in the amino acid sequencerepresented by SEQ ID NO:63 (name of antibody: Old17)(d) a heavy chain variable region comprising an amino acid sequence fromglutamine (Q) at position 20 to serine (S) at position 139 in the aminoacid sequence represented by SEQ ID NO:65 and a light chain variableregion comprising an amino acid sequence from aspartic acid (D) atposition 23 to lysine (K) at position 129 in the amino acid sequencerepresented by SEQ ID NO:67 (name of antibody: Old19)(e) a heavy chain variable region comprising an amino acid sequence fromglutamine (Q) at position 20 to serine (S) at position 138 in the aminoacid sequence represented by SEQ ID NO:69 and a light chain variableregion comprising an amino acid sequence from aspartic acid (D) atposition 23 to lysine (K) at position 129 in the amino acid sequencerepresented by SEQ ID NO:71 (name of antibody: New102) and(f) a heavy chain variable region and/or light chain variable region,which comprise amino acid sequences in which 1 to 3 amino acid residuesare deleted, substituted, added or inserted in the heavy chain variableregion and/or light chain variable region shown by (a) to (e).

(Antibody)

The antibody is used in a most broad sense and includes a monoclonalantibody, a polyclonal antibody, a multivalent antibody, a multispecificantibody (e.g., bispecific antibody) and also antibody fragments as longas these exhibit the desired biological activity.

The antibody contains a mature heavy chain or light chain variableregion sequence. In addition, the antibody also includes a modified formand variant form such as substitutions within or outside of a constantregion, a complementary determining region (CDR) or a framework (FR)region antibody of a mature heavy or light chain variable regionsequence of the antibody, and the like. In a specific embodiment, thesubstitution includes a conservative amino acid substitution is includedin the substitution.

In addition, the antibody also includes a subsequence of the matureheavy chain or light chain variable region sequence. In a specificembodiment, the subsequence is selected from Fab, Fab′, F(ab′)₂, Fv, Fd,single chain Fv (scFv), disulfide bond Fv (sdFv) and VL or VH.

In addition, the antibody also includes a heterogeneous domain. In aspecific embodiment, the heterogeneous domain includes a tag, adetectable label or a cytotoxic agent.

Examples of the antibody include a monoclonal antibody and a polyclonalantibody and any isotype or subclass thereof. In a specific embodiment,the aforementioned antibody is an isotype of IgG (e.g., IgG1, IgG2, IgG3or IgG4), IgA, IgM, IgE or IgD. The “monoclonal” antibody means anantibody that is based upon, obtained from a single clone including aeukaryote clone, a prokaryote clone or a phage clone or derived from asingle clone including a eukaryote clone, a prokaryote clone or a phageclone, based on a single clone including a eukaryote clone, a prokaryoteclone or a phage clone. Accordingly, the “monoclonal” antibody is astructurally defined substance and not a method by which it is produced.

The IL-3Rα antibody, anti-IL-3Rα and anti-IL-3Rα antibody mean anantibody which specifically binds to IL-3Rα. The specific binding meansthat it is selective for the epitope presenting in IL-3Rα. The specificbinding can be distinguished from non-specific binding using a knownassay in the technical field (e.g., immunoprecipitation, ELISA, flowcytometry, Western blotting).

When all or a part of antigen epitopes to which an IL-3Rα antibodyspecifically binds are present in different proteins, there is apossibility that this antibody can bind to the different proteins.Therefore, there is a possibility that the IL-3Rα antibody specificallybinds to other protein having high sequence or structural homology toIL-3Rα epitope depending on the sequence or structural homology toIL-3Rα epitope. Accordingly, there is a possibility that IL-3Rα antibodybinds to a different protein when an epitope having sufficient sequenceor structural homology is present in the different protein.

The IL-3Rα antibody includes isolated and purified antibodies. Theantibody of the invention including an isolated or purified IL-3Rαantibody includes human.

The term “(be) isolated” to be used as a modifier of a composition meansthat the composition is prepared by the hand of man or separated fromone or more other components in in vivo environment presenting in naturegenerally by one or more manipulative steps or processes. In general, acomposition separated in this manner does not substantially contain oneor more materials with which they normally associate in nature, such asone or more proteins, nucleic acids, lipids, carbohydrates and cellmembranes. Because of this, the isolated composition is separated fromother biological components in the cells of the organism in which thecomposition naturally occurs, or from the artificial medium in which itis produced (e.g., by synthesis or cell culture). For example, anisolated IL-3Rα antibody can be obtained from an animal in which theantibody is produced (e.g., non-transgenic mammals or transgenic mammals(rodents (mouse) or the ungulates (cattle)) and is separated from otherpolypeptides and nucleic acids. Accordingly, it is considered that theserum containing an antibody obtained from such an animal is isolated.The term “(be) isolated” does not exclude alternative physical forms,and for example, an isolated antibody could include antibodysubsequences and chimeras, multimers or derivatized forms.

The term “(be) purified” to be used as a modifier of a compositionrefers to a composition which is free of most of or substantially all ofthe materials with which it typically associates in nature. In general,a purified antibody is obtained from the components generally presentingin the antibody environment. Because of this, it is considered that anantibody supernatant which is separated from a cell culture mixture ofan antibody producing hybridoma is purified. Accordingly, the “(be)purified” does not require absolute purity and is context specific.Furthermore, the “(be) purified” composition can be combined with one ormore other molecules. Because of this, the term “(be) purified” does notexclude combination of composition. The purity can be determined by anoptional appropriate method such as UV spectrometry, chromatography(e.g., HPLC, gas phase), gel electrophoresis (e.g., silver or Coomassiestaining), sequence analysis (peptide and nucleic acid) and the like.

The “(be) purified” protein and nucleic acid include a protein and anucleic acid which are obtained by a standard purification method. Also,a protein and a nucleic acid obtained by recombination expression in ahost cell and chemical synthesis are also included in this term. Inaddition, the “(be) purified” can also refer to a composition in whichthe level of contaminants is lower than the level which is acceptable toa regulatory agency for administration to human or non-human animals,such as the Food and Drug Administration (FDA).

The IL-3Rα antibody includes an antibody which binds to IL-3Rα andmodulates function or activity of IL-3Rα in vivo or in vitro (e.g., in asubject). In the specification, the “to modulate” and the grammaticalvariations thereof when used in relation to the activity or function ofIL-3Rα mean that the IL-3Rα activity or function is detectably affected,modified or altered but does not include inhibition of IL-3 signaling.Accordingly, the IL-3Rα antibody which modulates the activity orfunction of IL-3Rα is an antibody that provides influence, modificationor alteration such that one or more of the IL-3Rα activity or functioncan be detected without inhibiting IL-3 signaling, and such an activityor function of IL-3Rα can includes, for example, binding of IL-3Rα withan IL-3Rα ligand (e.g., IL-3), an IL-3Rα-mediated signal transfer or anIL-3Rα-mediated cell response or a cell response that can be modulatedby IL-3Rα, or the activity or function of other IL-3Rα described in thespecification or, otherwise, is commonly known or can be known.

Examples of various non-limited IL-3Rα activities and functions whichcan be modulated include IL-3Rα mediated signal transduction or IL-3Rαmediated cellular response, cellular response which can be modulated viaIL-3Rα, cell proliferation or cell expansion (e.g., AML cell, CML cell,ALL cell, CLL cell, MDS cell, MM cell, SM cell, various lymphoma cells,monocytes, macrophages, mast cells, basophils, helper T cells,regulatory T cells, natural killer cells, myeloid progenitor cells andlymphoid progenitor cells), cell survival or apoptosis (e.g., AML cell,CML cell, ALL cell, CLL cell, MDS cell, MM cell, SM cell, variouslymphoma cells, monocytes, macrophages, mast cells, basophils, helper Tcells, regulatory T cells, natural killer cells, myeloid progenitorcells and lymphoid progenitor cells), cytokines (e.g., Th1, Th2 andnon-Th1/Th2 cytokines) and interferon expression or production,expression or production of anti-apoptosis protein or proapoptosisprotein, treatment, suppression or improvement of disorder, disease,physiological condition, pathological condition and symptom. Specificcytokines to be modulated are not limited and examples include IL-1,IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-14, IL-16, IL-17, IL-23, IL-26,TNF-a, and interferon γ (in vitro or in vivo). Specific anti-apoptosisproteins and proapoptosis proteins are not limited and examples includeBcl-xL, Bcl-2, Bad, Bim, and Mcl-1.

Therefore, examples of anti-IL-3Rα antibody described in the presentspecification include an antibody which modulates IL-3Rα mediated signaltransduction or IL-3Rα mediated cellular response, cellular responsewhich can be modulated via IL-3Rα, cell proliferation or cell growth(e.g., AML cell, CML cell, ALL cell, CLL cell, MDS cell, MM cell, SMcell, various lymphoma cells, monocytes, macrophages, mast cells,basophils, helper T cells, regulatory T cells, natural killer cells,myeloid progenitor cells and lymphoid progenitor cells), cell survivalor apoptosis (e.g., AML cell, CML cell, ALL cell, CLL cell, MDS cell, MMcell, SM cell, various lymphoma cells, monocytes, macrophages, mastcells, basophils, helper T cells, regulatory T cells, natural killercells, myeloid progenitor cells and lymphoid progenitor cells),cytokines (e.g., Th1, Th2 and non-Th1/Th2 cytokines) and interferonexpression or production, expression or production of anti-apoptosisprotein or proapoptosis protein, treatment, suppression or improvementof disorder, disease, physiological condition, pathological conditionand symptom. In the specific embodiments, anti-IL-3Rα antibody of thepresent invention can modulate expansion or survival of AML cell, numberof other blood cancer cell (e.g., CML cell, ALL cell, MDS cell, MM cell,SM cell or various lymphoma cell), growth or survival of non-cancerblood cell such as monocytes, macrophages, mast cells, basophils, helperT cells, regulatory T cells, natural killer cells, myeloid progenitorcells and lymphoid progenitor cells, and reduces, disappears or depletesAML cell, CML cell, ALL cell, CLL cell, MDS cell, MM cell, SM cell, orvarious lymphoma cells.

The IL-3Rα antibody includes a modified form such as a substitutionproduct (e.g., an amino acid substitution product) which is also calledas “variant”, an addition product, deletion product (e.g., a subsequenceor fragment) and the like. Such modified antibody forms and variantsretain at least partial function or activity of the IL-3Rα antibodyshown by the invention, such as binding with IL-3Rα, or modulation ofactivity or function (e.g., IL-3Rα signal transfer) of IL-3Rα.Accordingly, the modified IL-3Rα antibody can retain the ability tomodulate, for example, at least partial of IL-3Rα binding or one or moreof the IL-3Rα functions or activities (e.g., signal transfer, cellresponse and the like).

According to this specification, the term “to alter” (“to modify”) andthe grammatical variations thereof means that the compositionderivarates a reference composition. The modified proteins, nucleicacids and other compositions can have higher or lower activities than areference unmodified protein, nucleic acid or other composition or canhave a different function from a reference unmodified protein, nucleicacid or other composition.

Such an antibody containing an amino acid substitution can be encoded bynucleic acid. Accordingly, the present invention also provides anucleotide sequence encoding an antibody containing an amino acidsubstitution.

The term “identity” or “identical” means that two or more referencedsubstances are the same. Accordingly, when two protein sequences (e.g.,IL-3Rα antibodies) are identical, they have the same amino acidsequences at least within the referenced regions or portion. The term“identical region” means an identical region of two or more referencedsubstances. Thus, when two protein sequences are identical over one ormore sequence regions, they have identity within the regions.“Substantial identity” means that a molecule is structurally orfunctionally conserved such that the molecule has or is predicted tohave at least partial function or activity of one or more of referencemolecule functions or activities or relevant/corresponding region or aportion of the reference molecule to which it shares identity. Thus,polypeptides having substantial identity (e.g., IL-3Rα antibodies) haveor are predicted to have at least a part of the activity or function asa referenced polypeptide (e.g., IL-3Rα antibody). For example, in aspecific embodiment, it is considered that an IL-3Rα antibody having oneor more modifications (e.g., deletion, substitution, addition orinsertion of 1 to 3 amino acid residues) which retain at least partialactivity or function of the unmodified IL-3Rα antibody has substantialidentity to the reference IL-3Rα antibody.

Due to variations between structurally related protein and functionallyrelated protein, the amount of sequence identity required to retainfunctions or activity on the protein, region and function or activity ofthe region. In the case of protein, an activity or function can beretained by the presence of merely 30% of amino acid sequence identity,but in general, higher identity of 50%, 60%, 75%, 85%, 90%, 95%, 96%,97% or 98%, to the reference sequence is present. The extent of identitybetween two sequences can be verified using a computer program ormathematic algorithm conventionally known in the technical field. Insuch an algorithm which calculates ratio of sequence identity(homology), in general, sequence gaps and mismatches over the comparisonregion are accounted. For example, BLAST (e.g., BLAST 2.0) retrievalalgorithm (e.g., see Altschul et al., J. Mol. Biol., 215: 403 (1990),publicly available through NCBI) has the following illustrativeretrieval parameters: mismatch −2; gap start 5; gap elongation 2. In thepolypeptide sequence comparison, the BLASTP algorithm is typically usedin combination with a scoring matrix such as PAM 100, PAM 250, BLOSUM62, BLOSUM50.FASTA (e.g., FASTA 2 and FASTA 3) and the like, and SSEARCHsequence comparison program is also used for determining the extent ofidentity (Pearson et al., Proc. Natl. Acad. Sci. USA, 85: 2444 (1988);Pearson, Methods Mol. Bio., 132: 185 (2000); and Smith et al., J. Mol.Biol., 147: 195 (1981)). A program has also been developed fordetermining protein structural similarity using topological mappingbased on Delaunary (Bostick et al., Biochem. Biophys. Res. Commun., 304:320 (2003)).

A “conservative substitution” is a substitution of one amino acid by abiologically, chemically or structurally similar residue. Biologicalsimilarity means that a biological activity such as IL-3Rα bindingactivity is not destroyed by the substitution. Structural similaritymeans that amino acids have side chain with similar length (e.g.,alanine, glycine and serine) or have similar size. Chemical similaritymeans that the residues have the same charge or are hydrophilic orhydrophobic. Specific examples include substitution of one hydrophobicresidue such as isoleucine, valine, leucine, and methionine with otherresidue, or the substitution of one polar residue with other residuesuch as the substitution of arginine with lysine, the substitution ofglutamic acid with aspartic acid, or the substitution of glutamine withasparagine, and the substitution of serine with threonine.

In addition, examples of the modified antibody include peptide mimeticshaving one or more D-amino acids substituted with L-amino acids (and amixture thereof), structural and functional analogs such as synthesizedor non-natural amino acids or amino acid analogs, and derivatized formthereof. Examples of modification include a cyclic structure such as anend-to-end amide bond between the amino and carboxy-terminus of themolecule or intra- or inter-molecular disulfide bond or intramolecularor intermolecular disulfide bond.

Additional non-limiting specific examples of the amino acidmodifications include partial sequence (subsequence) and fragment ofIL-3Rα. Exemplary subsequence and fragment of IL-3Rα include a part ofthe IL-3Rα sequence to which the exemplary IL-3Rα antibody of theinvention binds. Also, the exemplary subsequence and fragment of IL-3Rαinclude an immunogenicity region such as a part of the IL-3Rα to whichthe exemplary IL-3Rα antibody of the invention binds.

According to the invention, there is provided a nucleic acid encoding anIL-3Rα antibody subsequence of fragment which retains at least a part ofthe function or activity of the IL-3Rα antibody and an unmodified orreference IL-3Rα antibody. In this specification, the term “subsequence”or “fragment” means a portion of a full length molecule. The amino acidsequence encoding the subsequence of the IL-3Rα antibody has amino acidsof smaller than those of the full length IL-3Rα antibody by at least one(e.g., deletion of one or more inner or terminal amino acids from theamino terminus or carboxy terminus). The subsequence of IL-3Rα antibodyhas amino acids of smaller than those of the full length IL-3Rα antibodyby at least one. The nucleic acid subsequence has nucleotides of smallerthan those of the full length comparative nucleic acid sequence by atleast one. Accordingly, the subsequence can be an optional length withinthe full length of native IL-3Rα.

The IL-3Rα antibody subsequence and fragment can have a binding affinityas the full length antibody, a binding specificity as the full lengthantibody or one or more activities or functions as the full lengthantibody, such as the function or activity of an IL-3Rα antagonist oragonist antibody. The terms “functional subsequence” and “functionalfragment” in the case of referring to the antibody mean an antibodyportion which retains one or more functions or activities as the fulllength reference antibody, such as at least a part of the function oractivity of IL-3Rα antibody. For example, an antibody subsequence whichbinds to IL-3Rα or a fragment of IL-3Rα is considered a functionalsubsequence.

The antibody subsequence and fragment can be combined. For example, a VLor VH subsequence can be connected by a linker sequence and thereby canform a VL-VH chimeric body. A combination of single chain Fv(scFv)subsequences can be connected by a linker sequence and thereby can forma scFv-scFv-chimeric body. The IL-3Rα antibody subsequence and fragmentinclude a single chain antibody or variable region alone or incombination with all or a portion of other IL-3Rα antibody subsequence.

The antibody subsequence and fragment can be prepared by hydrolysis ofthe antibody by its proteolysis for example by a pepsin or papaindigestion of the whole antibody. The antibody subsequence and fragmentobtained by enzymatic cleavage with pepsin provide a 5S fragmentrepresented by F(ab′)₂. This fragment can be further cleaved using athiol reducing agent to form a 3.5S Fab′ monovalent fragment.Alternatively, an enzymatic cleavage using pepsin directly produces twomonovalent Fab′ fragments and Fc fragment (see e.g., U.S. Pat. No.4,036,945 and U.S. Pat. No. 4,331,647; and Edelman et al., MethodsEnzymol., 1: 422 (1967)). Other methods of cleaving an antibody, such asseparation of heavy chain for forming a monovalent light chain-heavychain fragment, further cleavage of the fragment or other enzymatic orchemical method may be used.

A protein and an antibody, as well as subsequence thereof and fragmentcan be prepared using a genetic engineering. The technology includes thefull or partial gene encoding a protein or an antibody is expressed in ahost cell such as a COS cell and E. Coli. A recombinant host cellsynthesizes the full or subsequence such as scFv (such as Whitlow et al,In: Methods: A Companion to Methods in Enzymology 2:97 (1991), Bird etal, Science 242:423 (1988); and U.S. Pat. No. 4,946,778). A single chainFv and an antibody can be prepared in accordance with the procedure asdescribed in U.S. Pat. No. 4,946,778 and U.S. Pat. No. 5,258,498; Hustonet al, Methods Enzymol 203:46 (1991); Shu et al, Proc. Natl. Acad. Sci.USA 90:7995 (1993); and Skerra et al, Science 240:1038 (1988).

The modified form includes a derivatized sequence such as amino acids inwhich the free amino groups form amine hydrochloride, p-toluenesulfonylgroup and carbobenzoquinone group; the free carboxy groups which form asalt or methyl and ethyl ester; and the free hydroxyl groups form anO-acyl or O-alkyl derivative, and naturally existing amino acidderivatives such as 4-hydroxyproline (derivative of proline),5-hydroxylysine (derivative of lysine), homoserine (derivative ofserine), ornithine (derivative of lysine) and the like. The modificationcan be carried out using a method conventionally known in the technicalfield (e.g., site-specific deletion or insertion mutagenesis based onPCR, chemical modification and mutagenesis, crosslinking and the like).

Addition products and insertion products are included in the modifiedforms of protein (e.g., antibody), nucleic acid and other compositions.For example, the addition can be a covalent or non-covalent bond withany type of molecules of protein (e.g., antibody), nucleic acid or othercompositions. In general, addition and insertion confer differentfunction or activity.

Fusion (chimeric) polypeptides or nucleic acid sequences are included inthe addition product and insertion product, and these are sequenceshaving one or more molecules which are generally not present in thereference native (wild type) sequence covalently attached to theaforementioned sequence. A specific example is an amino acid sequence ofother protein (e.g., an antibody) for producing a multifunctionalprotein (e.g., a multispecific antibody).

Also, the antibody of the invention include a chimeric or fusion productin which one or more additional domains are covalently linked thereto inorder to confer a different or complementary function or activity.Examples of the antibody include a chimeric or fusion product which doesnot naturally present in natural and in which two or more amino acidsequences are mutually bonded.

According to the invention, there are provided an IL-3Rα antibody whichcontains a heterologous domain and a nucleic acid that encodes theIL-3Rα antibody. The heterologous domain can be an amino acid additionproduct or insertion product, but does not limited to an amino acidresidue. Accordingly, the heterologous domain can be composed of any oneof various different types of small or large functional parts. Such apart includes a nucleic acid, a peptide, a carbohydrate, a lipid orsmall organic compound such as a drug, a metal (gold, silver) and thelike.

Non-limiting specific examples of the heterologous domain include a tag,a detectable label and a cytotoxic agent. Specific examples of the tagand detectable label include T7-, His-, myc-, HA- and FLAG-tags; enzymes(horseradish peroxidase, urease, catalase, alkaline phosphatase,β-galactosidase, chloramphenicol transferase); enzyme substrates;ligands (e.g., biotin); receptors (avidin); radionuclide (e.g., C14,S35, P32, P33, H3, I125 and I131); electron density reagents; energytransfer molecules; paramagnetic labels; fluorophore (fluorescein,Rhodamine, Phycoerythrin); chromophore; chemiluminescence agents(imidazole, luciferase) and bioluminescence agents. Specific examples ofthe cytotoxic agent include diphtheria toxin (diphtheria, toxin),cholera toxin and lysine.

A linker sequence may be inserted between the protein (e.g., anantibody), nucleic acid or other composition and the addition product orinsertion product (e.g., a heterologous domain) so that the twosubstances maintain at least a part of different function or activity.The linker sequence may have one or more properties which can accelerateeither of the domains or can carry out mutual reaction with either ofthe domains, and such characteristics include impossibility to form aflexible structure and an ordered secondary structure or hydrophobicproperty or charging property. Examples of the amino acids which aregenerally found in the flexible protein regions include glycine,asparagine and serine. Other amino acids close to neutral such asthreonine and alanine may also be used in the linker sequence. Thelength of the linker sequence can be varied (e.g., see U.S. Pat. No.6,087,329). The linker further include chemical crosslinking agents andbinding agents (conjugating agents) such as a sulfo-succinimidylderivative (sulfo-SMCC, sulfo-SMPB), disuccinimidyl suberate (DSS),disuccinimidyl glutarate (DSG) and disuccinimidyl tartarate (DST).

Further examples of the addition include any one of glycosylation, fattyacid, lipid, acetylation, phosphorylation, amidation, formylation,ubiquitination and derivatiation by a protecting or blocking group and alarge number of chemical modifications. Other substitutions andpossibilities can be easily understood by those skilled in the art andare considered to be within the scope of the invention.

Such a modified sequence can be prepared using recombinant DNAtechniques which mediate cell expression or in vitro translation.Polypeptides and nucleic acid sequences can also be prepared by aconventionally known method in the technical field such as chemicalsynthesis using an automatic peptide synthesizer (see e.g., AppliedBiosystems, Foster City Calif.).

Modified and variant antibodies such as substitution products,subsequences addition products and the like can maintain detectableactivity of IL-3Rα antibody. In an embodiment, the modified antibody hasthe activity to bind to IL-3Rα molecule and induces reduction orelimination of IL-3Rα expression cells by an immune system mainlycentering on an effector cell. The modified antibody relates to thefunctional control of IL-3Rα expression cells and induces survival,growth, resting, cell death and the like of the cells. The cell deathincludes apoptosis, necrosis, autophagy and the like.

(Screening Method of IL-3Rα)

According to the invention, there are further provided a cell-freemethod and a cell-based method (e.g., in vivo or in vitro) which screen,detect and identify IL-3Rα (e.g., in a solution or by a solid phase).These methods can be carried out in a solution in vitro using abiomaterial or sample, and in vivo for example using a sample of ananimal-derived cell (e.g., lymphocyte). In an embodiment, the methodcomprises a step of contacting a biomaterial or sample with an antibodybound to IL-3Rα under a condition of allowing binding of the antibodywith IL-3Rα and a step of assaying for the antibody bound to IL-3Rα. Thepresence of IL-3Rα is detected by binding of the antibody to bind toIL-3Rα. In an embodiment, IL-3Rα is present in a cell or tissue. Inanother embodiment, the aforementioned biomaterial or sample is obtainedfrom a mammal analyte.

The term “contacting” when it is used in relation to the compositionsuch a protein (e.g., IL-3Rα antibody), a material, a sample ortreatment means a direct or indirect interaction between the composition(e.g., IL-3Rα antibody) and other referenced substance. Specificexamples of the direct interaction include bonding. Specific examples ofthe indirect interaction include a case in which the composition actsupon an intermediate molecule and this intermediate molecule then actsupon the referenced substance. Accordingly, for example, contacting acell (e.g., lymphocyte) to IL-3Rα antibody includes to allow theantibody to bind to the cell (e.g., through binding to IL-3Rα) or toallow the antibody to act on an intermediate substance, followed by theaction of this intermediate substance upon the cell.

The terms “assaying” and “measuring” and grammatical variations thereofare synonymously used in the specification and mean either ofqualitative measurement and quantitative measurement or both ofqualitative measurement and quantitative measurement. When these termsare used in relation to binding, they include any means of evaluatingrelative amount, affinity or specificity of binding including variousmethods which are described in the specification and conventionallyknown in the technical field. For example, binding of the IL-3Rαantibody with IL-3Rα can be assayed or measured by a flow cytometryassay.

(Production of Antibody)

The invention also provides a method for producing a human IL-3Rαantibody having cytotoxicity for IL-3Rα positive cells. In anembodiment, the method comprises administering a human IL-3Rαextracellular region conjugated with a human IL-3Rα recombinant proteinor an IL-3Rα gene introduced cell into animals capable of expressinghuman immunoglobulin (e.g., transgenic mice or transgenic cattle);screening the animal for expression of a human IL-3Rα antibody;selecting the animal producing the human IL-3Rα antibody; and isolatingthe antibody from the selected animal.

The IL-3Rα protein suitable for the antibody production can be producedby any one of various standard protein purification and recombinantexpression techniques. For example, the IL-3Rα sequence can be preparedby standard peptide synthesis techniques such as a solid phasesynthesis. In order to facilitate purification of the expressed orsynthesized protein, a portion of the protein may contain an amino acidsequence such as a FLAG tag, a T7 tag, a polyhistidine sequence or thelike. The protein is expressed inside the cells and can be purified. Theprotein can be expressed by a recombination method as a part of afurther large protein (e.g., a fusion or chimeric product). Theembodiment of the IL-3Rα suitable for generating immune responseincludes IL-3Rα subsequences such as an immunogenicity fragment. Furtherembodiment of IL-3Rα includes an IL-3Rα expressing cell, an IL-3Rαcontaining preparation or cell extract or fraction and a partiallypurified IL-3Rα.

The method for preparing polyclonal antibody and monoclonal antibody isconventionally known in the technical field. For example, IL-3Rα or itsimmunogenicity fragment used for immunizing an animal by optionallyconjugating with a carrier such as keyhole limpet hemocyanin (KLH) orovalbumin (e.g., BSA) or mixing with an adjuvant such as completeFreund's adjuvant or incomplete Freund's adjuvant. By isolating a spleencell derived from an immunized animal which responds to IL-3Rα, it canbe fused with myeloma cell using hybridoma techniques. The monoclonalantibodies produced by hybridomas can be screened for reactivity withIL-3Rα or immunogenicity fragment thereof.

The animal which can be immunized includes the primates, mouse, rat,rabbit, goat, sheep, cattle and guinea pig. The initial and anyoptionally subsequent immunization may be by intravenous route,intraperitoneal route, intramuscular route or subcutaneous route.Further, in order to increase immune response, the antigen can beconjugated with other protein such as keyhole limpet hemocyanin (KLH),thyroglobulin and tetanus toxoid, or can be mixed with an adjuvant suchas complete Freund's adjuvant, incomplete Freund's adjuvant and thelike. The initial and any optionally subsequence immunization may bethrough intraperitoneal route, intramuscular route, intraocular route orsubcutaneous route. The immunization may be at the same concentration ordifferent concentration of an IL-3Rα preparation or at regular orirregular intervals.

The animal includes those which are genetically modified to includehuman gene loci, and a human antibody can be prepared using the same.Examples of the transgenic animals with one or more human immunoglobulingenes, are described for example in U.S. Pat. No. 5,939,598, WO02/43478and WO02/092812. Using conventional hybridoma technique, an spleen cellswhich are isolated from immunized mouse having high responders to theantigen and are fused with myeloma cell. A monoclonal antibody whichbinds to IL-3Rα can be obtained.

The method for producing a human polyclonal antibody and a humanmonoclonal antibody is further described (see, such as Kuroiwa et al,Nat. Biotechnol. 20:889 (2002); WO98/24893; WO92/01047; WO96/34096;WO96/33735; U.S. Pat. No. 5,413,923; U.S. Pat. No. 5,625,126; U.S. Pat.No. 5,633,425; U.S. Pat. No. 5,569,825; U.S. Pat. No. 5,661,016; U.S.Pat. No. 5,545,806; U.S. Pat. No. 5,814,318; U.S. Pat. No. 5,885,793;U.S. Pat. No. 5,916,771; and U.S. Pat. No. 5,939,598).

The term “human” when it is used in reference to an antibody means thatamino acid sequence of the antibody is completely the human amino acidsequence, namely is human heavy chain and human light chain variableregions and human constant region. Accordingly, all of the amino acidsare human amino acids or present in the human antibody. An antibodywhich is a non-human antibody can be made into a complete human antibodyby substituting the non-human amino acid residues with the amino acidresidues which are present in the human antibody. The amino acidresidues which are present in the human antibody, CDR region map andhuman antibody consensus residues are well known in the technical field(see e.g., Kabat, Sequences of Proteins of Immunological Interest,4^(th) edition, US Department of Health and Human Services, PublicHealth Service (1987); Chothia and Lesk (1987)). A consensus sequence ofhuman VH subgroup III based on the investigation carried out using 22known human VH III sequences as the object and a consensus sequence ofhuman VL κ chain subgroup I based on the investigation carried out using30 known human κ chain I sequences as the object are described inPadlan, Mol. Immunol., 31: 169 (1994) and Padlan, Mol. Immunol., 28: 489(1991). Accordingly, the human antibody includes an antibody in whichone or more amino acid residues have been substituted with one or moreamino acids existing in an optional other human antibody.

Examples of the anti-IL-3Rα antibody include antibodies prepared using aknown method in the technical field, such as CDR-grafting (EP 239,400;WO91/09967; U.S. Pat. No. 5,225,539; U.S. Pat. No. 5,530,101; and U.S.Pat. No. 5,585,089), veneering, resurfacing (EP592,106; EP519,596;Padlan, Molecular Immunol. 28: 489 (1991); Studnicka et al., ProteinEngineering 7: 805 (1994); Roguska et al., Proc. Nat'l Acad. Sci. USA91: 969 (1994)) and chain shuffling (U.S. Pat. No. 5,565,332). In orderto produce a humanized antibody, human consensus sequence (Padlan, Mol.Immunol. 31:169 (1994); and Padlan, Mol. Immunol. 28: 489 (1991)) hasbeen used (Carter et al., Proc. Natl. Acad. Sci. USA 89: 4285 (1992);and Presta et al, J. Immunol. 151: 2623 (1993)).

The term “humanized” when it is used in relation to an antibody meansthat amino acid sequence of the antibody has one or more non-human aminoacid residues (e.g., mouse, rat, goat, rabbit and the like) ofcomplement determining region (CDR) which specifically binds to adesired antigen in an acceptor human immunoglobulin molecule and one ormore human amino acid residues (amino acid residues which are flankedwith CDR) in Fv framework region (FR). The antibody called “primatized”is within the scope of meaning of “humanized”, except that amino acidresidues of the acceptor human immunoglobulin molecule and frameworkregion can be any primate amino acid residues (e.g., monkey, gibbon,gorilla, chimpanzee, orangutan, macaque monkey) in addition to any humanresidues. Human FR residues of immunoglobulin can be substituted withcorresponding non-human residues. Accordingly, for example, in order toalter, generally to improve, antigen affinity or specificity, residuesin the CDR or human framework region can be substituted withcorresponding residues from the non-human CDR or framework region donorantibody. The humanized antibody can contain residues which cannot befound in the human antibody and donor CDR or framework sequence. Forexample, it can be predicted that FR substitution at a particularposition which cannot be found in human antibody or donor non-humanantibody can improve binding affinity or specific human antibody at thisposition. Antibody framework and CDR substitutions based on themolecular modeling are conventionally known in the technical field, forexample by the modeling of interaction of CDR and framework residues toidentify framework residues important for antigen binding and thesequence comparison for identifying unusual framework residues at thespecific position (see e.g., U.S. Pat. No. 5,585,089; and Riechmann etal., Nature, 332:323 (1988)).

Chimeric antibodies are included in the IL-3Rα antibody. According tothis specification, the term “chimeric” and the grammatical variationsthereof when it is used in relation to antibodies mean that amino acidsequence of the antibody contains one or more portion which is derivedfrom two or more different species, is obtained or isolated from two ormore different species or is based on two or more different species. Forexample, a portion of the antibody can be human (e.g., constant region)and other portion of the antibody can be non-human (e.g., a mouse heavychain or a mouse light variable region). Accordingly, an example of thechimeric antibody includes an antibody in which the different portion ofthe antibody is derived from a different species. Different from thehumanized or primatized antibody, the chimeric antibody can have asequence of different species in an arbitrary region of the antibody.

The method for producing a chimeric antibody is known in the technicalfield (such as Morrison, Science 229: 1202 (1985); Oi et al.,BioTechniques 4: 214 (1986); Gillies et al., J. Immunol. Methods 125:191 (1989); U.S. Pat. No. 5,807,715; U.S. Pat. No. 4,816,567; and U.S.Pat. No. 4,816,397). For example, in Munro, Nature 312: 597 (1984);Neuberger et al., Nature 312: 604 (1984); Sharon et al., Nature 309: 364(1984); Morrison et al., Proc. Nat'l. Acad. Sci. USA 81: 6851 (1984);Boulianne et al., Nature 312: 643 (1984); Capon et al, Nature 337: 525(1989); and Traunecker et al., Nature 339: 68 (1989), a chimericantibody in which a variable region of antibody derived from one speciesis replaced by a variable region of antibody derived from anotherspecies.

In addition, the anti-IL-3Rα antibody can be prepared by hybridomatechnique, recombinant technique, and phage display technique, and acombination thereof (see U.S. Pat. No. 4,902,614, U.S. Pat. No.4,543,439, and U.S. Pat. No. 4,411,993; and also see MonoclonalAntibodies. Hybridomas: A New Dimensionin Biological Analyses, PlenumPress, Kennett, McKearn, and Bechtol et al, 1980, and Harlow et al.,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,the second edition, 1988).

The human anti-human IL-3Rα antibody of the invention was produced usingchromosome-transferred mice (KM mice (trademark)) immunized with variousforms of soluble form of recombinant human IL-3Rα proteins or cell linesexpressing IL-3Rα (WO02/43478, WO02/092812, and Ishida et al., IBC's11^(th) Antibody Engineering Meeting, Abstract (2000)). Since the humananti-human IL-3Rα antibody detectably stains not a non-transformedparent cell line but a human IL-3Rα stable transfectant cell line, suchas Jurkat-IL-3Rα cell and L929-IL-3Rα cell, the antibody specifically isshown to bind to human IL-3Rα.

The antibody of the invention can have κ light chain sequence or λ lightchain sequence, full length of either one of them as present innaturally existing antibody, a mixture thereof (namely a fusion productof κ chain sequence and λ chain sequence) and subsequences/fragmentsthereof. The naturally presenting antibody molecules contain two κ lightchains or two λ light chains.

The invention provides a method for preparing an antibody whichspecifically binds to IL-3Rα. In a specific embodiment, the method forpreparing IL-3Rα antibody comprises administering human IL-3Rα, asubsequence thereof or a fragment thereof (e.g., IL-3Rα extracellularregion), conjugated with a human Fc recombinant protein if necessary, toanimals which can express human immunoglobulin (e.g., transgenic mice ortransgenic cattle), screening the animals for their expression of humanIL-3Rα antibody, selecting an animal which produces human IL-3Rαantibody and isolating the antibody from the selected animal. In anembodiment, whether or not the human IL-3Rα antibody has an IL-3Rαantagonist or agonist activity is judged by this method.

The effector activity means an antibody-dependent activity induced viaFc region of antibody, and such as antibody-dependent cellularcytotoxicity (ADCC activity), complement-dependent cytotoxicity (CDCactivity), antibody-dependent phagocytosis (ADP activity) by phagocytessuch as macrophage and dendritic cell, and the like, are known.

As a method for controlling effector activity of the anti-IL-3Rαmonoclonal antibody of the invention, examples include a method whichcontrols the amount of the fucose (also called core fucose) which isbound to N-acetylglucosamine (GlcNAc) through α-1,6 bond in a reducingend of a complex-type N-linked sugar chain which is bound to asparagine(Asn) at position 297 of an Fc region of an antibody (WO2005/035586,WO2002/31140, and WO00/61739), a method in which is controlled bymodifying amino acid residues of Fc region of the antibody, and thelike. The effector activity can be controlled by applying any one ofthese methods to the anti-IL-3Rα monoclonal antibody of the invention.

By controlling the content of the core fucose of complex-type N-linkedsugar chain of Fc of the antibody, effector activity of the antibody canbe increased or decreased. As a method for reducing the content of thefucose which binds to the complex-type N-linked sugar chain which isbound to Fc of the antibody, defucosylation (defucosylated ornon-fucosylated) can be mentioned. The defucosylation is to express anantibody using CHO cell from which α1,6-fucosyltransferase gene isdeleted, and an antibody to which fucose is not bound can be obtained.The antibody to which fucose is not bound has high ADCC activity. On theother hand, as a method for increasing the content of the fucose whichbinds to the complex-type N-linked sugar chain to which Fc of theantibody is bound, the antibody to which fucose is bound can be obtainedby expressing the antibody using a host cell in whichα1,6-fucosyltransferase gene is introduced. The antibody to which fucoseis bound has the ADCC activity lower than that of the antibody to whichfucose is not bound.

In addition, ADCC activity and CDC activity can be increased ordecreased by modifying amino acid residues of the Fc region of theantibody. For example, CDC activity of the antibody can be increased byusing the amino acid sequence of the Fc region described in US2007/0148165. Also, ADCC activity or CDC activity can be increased ordecreased by carrying out the amino acid modification described in U.S.Pat. No. 6,737,056, U.S. Pat. No. 7,297,775 and U.S. Pat. No. 7,317,091.Further, an antibody in which effector activity of the antibody iscontrolled can be obtained by using the above-mentioned methods incombination in one antibody.

According to the invention, the nucleotide sequence of the inventionsuch as of a vector and the like is further provided. In an embodiment,the vector comprises a nucleic acid sequence encoding an IL-3Rα antibodyor a subsequence or fragment thereof.

The nucleic acid can have various lengths. The length of the nucleicacid encoding the IL-3Rα antibody of the present invention or thesubsequence thereof is generally about 100 to 600 nucleotides, or anynumerical value or range within encompassing such lengths the abovedescribed range; 100 to 150, 150 to 200, 200 to 250, 250 to 300, 300 to350, 350 to 400, 400 to 450, 450 to 500, 500 to 550 or 550 to 600nucleotide length, or any numerical value or range or value within orencompassing such length the above described range. Examples of thelength of nucleic acid encoding the IL-3Rα antibody of the presentinvention or the subsequence thereof include generally 10 to 20, 20 to30, 30 to 50, 50 to 100, 100 to 150, 150 to 200, 200 to 250, 250 to 300,300 to 400, 400 to 500, 500 to 600 nucleotides and any numerical valueor range within or encompassing such length.

The terms “nucleic acid” and “polynucleotide” means at least two or moreribo- or deoxy-ribo nucleic acid base pairs (nucleotide) linked whichare through a phosphoester bond or equivalent. The nucleic acid includespolynucleotide and polynucleoside. The nucleic acid includes a singlemolecule, a double molecule, a triple molecule, a circular molecule or alinear molecule. Examples of the nucleic acid include RNA, DNA, cDNA, agenomic nucleic acid, a naturally existing nucleic acid and anon-natural nucleic acid such as a synthetic nucleic acid, but are notlimited. Short nucleic acids and polynucleotides (e.g., 10 to 20, 20 to30, 30 to 50, 50 to 100 nucleotides) are commonly called“oligonucleotides” or “probes” of single-stranded or double-strandedDNA.

Nucleic acid can be prepared using various standard cloning techniquesand chemical synthesis techniques. Examples of the techniques includebut are not limited to, nucleic acid amplification such as polymerasechain reaction (PCR), with genomic DNA or cDNA targets using primers(e.g., a degenerate primer mixture) which can be annealed with anantibody encoding sequence. In addition, nucleic acid can also beprepared by chemical synthesis (e.g., solid phase phosphoamiditesynthesis) or transcription from a gene. Thereafter, the preparedsequence can be expressed by a cell (e.g., a host cell such as yeast,bacteria or eukaryote (an animal or mammal cell or in a plant)) afterthe sequence cloned into a plasmid and then amplified, or the sequenceis translated in vitro.

A vector is a vehicle which can be manipulated by insertion orincorporation of nucleic acid. Examples of the vector include a plasmidvector, a virus vector, a prokaryote (bacterium) vector and a eukaryote(plant, fungi, mammals) vector. The vector can be used for in vitro orin vivo expression of nucleic acid. Such a vector is called “expressionvector” and is useful for the transfer of nucleic acid including anucleic acid which encodes an IL-3Rα antibody or its subsequence orfragment and the expression of an encoded protein by in vitro (e.g., ina solution or on solid phase), by a cell or by in vivo in a subject.

In addition, the vector can also be used for manipulation of nucleicacids. For genetic manipulation, an inserted nucleic acid can betranscribed or translated using a “cloning vector” in vitro (e.g., in asolution or on solid phase), in a cell or in vivo in a subject.

In general, the vector contains an origin of replication foramplification in a cell in vitro or in vivo. Control elements such as anexpression control element present in the vector can be included inorder to facilitate transcription and translation, if necessary.

A vector can include a selection marker. The “selection marker” is agene which allows for the selection of a cell containing the gene.“Positive selection” means a process for selecting a cell containing theselection marker due to a positive selection. Drug resistance is anexample of the positive selection marker, and a cell containing themarker will survive in culture medium containing the drug and a cellwhich does not contain the marker will die. Examples of the selectionmarker include drug resistance genes such as neo which providesresistance to G418; hygr which provides resistance to hygromycin; purowhich provides resistance to puromycin, and the like. Other positiveselection maker includes genes which enable identification or screeningof a cell containing the marker. Examples of these genes include afluorescent protein (GFP and GFP-like chromophore, luciferase) gene,lacZ gene, alkaline phosphatase gene, and a surface marker such as CD8.“Negative selection” means a process for killing cells which containnegative selection markers by exposing to an appropriate negativeselection agent. For example, a cell containing a herpes simplex virusthymidine kinase (HSV-tk) gene (Wigler et al., Cell, 11: 223 (1977)) issensitive to a drug ganciclovir (GANC). Similarly, gpt gene makes a cellsensitive to 6-thioxantine.

The virus vector includes those which are based on retroviral (alentivirus for infecting not only dividing cells but also non-dividingcells), foamy virus (U.S. Pat. No. 5,624,820, U.S. Pat. No. 5,693,508,U.S. Pat. No. 5,665,577, U.S. Pat. No. 6,013,516 and U.S. Pat. No.5,674,703; WO 92/05266 and WO 92/14829), adenovirus (U.S. Pat. No.5,700,470, U.S. Pat. No. 5,731,172 and U.S. Pat. No. 5,928,944),adeno-associated virus (AAV) (U.S. Pat. No. 5,604,090), a herpes simplexvirus vector (U.S. Pat. No. 5,501,979), a cytomegalovirus (CMV) systemvector (U.S. Pat. No. 5,561,063), reovirus, rotavirus genome, simianvirus 40 (SV40) or papilloma virus (Cone et al., Proc. Natl. Acad. Sci.USA, 81:6349 (1984); Eukaryotic Viral Vectors, Cold Spring HarborLaboratory, edited by Gluzman, 1982; Sarver et al., Mol. Cell. Biol., 1:486 (1981); U.S. Pat. No. 5,719,054). Adenovirus efficiently infects aslowly replicating and/or terminally differentiated cell, and can beused to target the slowly replicating cell and/or terminallydifferentiated cell. Additional examples of virus vectors useful forexpression include parbovirus, Norwalk virus, corona virus, paramyxovirus and rhabdo virus, toga virus (e.g., Sindobis virus and Semlikiforest virus) and vesicular stomatitis virus (VSV).

A vector comprising a nucleotide acid can be expressed when the nucleicacid is connected to expression elements so as to function. The term“connected so as to function” (operably linked) means that a physical orfunctional relation between the elements referred to that permit them tooperate in their intended fashion. Accordingly, the nucleic acid“operably linked” to an expression control element means that thecontrol element modulates nucleic acid transcription and, asappropriate, translation of the transcription product.

The “expression control element” or “expression control sequence” is apolynucleotide which influences upon expression of an operably linkednucleic acid. Promoters and enhancers are non-limiting specific examplesof expression controlling elements and sequences. The “promoter” is acis-acting DNA regulatory region which can initiate transcription ofdownstream (3′ direction) nucleic acid sequence. A nucleotide whichaccelerates transcription initiation is included in the promotersequence. The enhancer also regulates nucleic acid expression but actsat a distance from the transcription initiation site of the nucleic acidto which it is operably linked. When the enhancer is present in eitherthe 5′ or 3′ end of the nucleic acid as well as within the nucleic acid(e.g., intron or coding sequence), the enhancer further functions.Additional examples of the expression control element include a leadersequence and a fusion partner sequence, an internal ribosome entry site(IRES) element for preparing multigene, or polycistronic message,splicing signal of intron, maintenance of correct reading frame of geneto enable inframe translation of mRNA, polyadenylation signal whichproduces proper polyadenylation of the transcription product ofinterest, and stop codons.

Examples the expression control element include a “constitutional”element in which transcription of an operably linked nucleic acid occurswithout the presence of signals or stimulus. The expression controlelement which confers expression in response to the signal or stimulusand increase or decrease expression of the operably linked nucleic acidis “adjustable”. The adjustable element which increases expression ofthe operably linked nucleic acid in response to a signal or stimulus iscalled “inducible element”. The adjustable element which decreasesexpression of the operably linked nucleic acid in response to a signalor stimulus is called “repressor element” (namely, the signal decreasesthe expression; and the expression increases when the signal is removedor not present).

Examples of the constitutional promoter for bacterial expression includean inducing promoter, such as T7 and pL, plac, ptrp and ptac (ptrp-lachybrid promoter) of bacteriophage λ and the like. For insect cellsystem, a constitutional or an inducible promoter (e.g., ecdysone) canbe used. The constitutional promoter for yeast include an inducingpromoter such as ADH, LEU2, GAL and the like (e.g., see Ausubel et al.,In: Current Protocols in Molecular Biology, Vol. 2, Chapter 13, GreenePublish. Assoc. & Wiley Interscience edition, 1988; Grant et al., In:Methods in Enzymology, 153: 516-544 (1987) Wu & Grossman, 1987, Acad.Press, N.Y.; Glover, DNA Cloning, Vol. 11, Chapter 3, IRL Press, Wash.,D.C., 1986; Bitter, In: Methods in Enzymology, 152: 673-684 (1987),edited by Berger & Kimmel, Acad. Press, N.Y.; and Strathern et al., TheMolecular Biology of the Yeast Saccharomyces, edited by Cold SpringHarbor Press, Vol. 1 and Vol. 11 (1982)).

For the expression in mammals, a constitutional promoter derived from avirus or other origin can be used. For example, inducible promotersderived from CMV, SV40, or a viral long terminal repeated sequence(LTR), or mammal cell genome (e.g., metallothionein 11A promoter; heatshock promoter, steroid/thyroid hormone/retinoic acid respondingelement) or mammal virus (e.g., adenovirus late promoter; mouse breastcancer virus LTR) can be used.

Examples of the expression control element include an element which isactive in a specific tissue or cell types, and such an element is called“tissue specific expression control element”. In general, the tissuespecific expression control element is more active in specific cells ortissue types, and this is because this tissue specific expressioncontrol element is recognized by a transcription activating proteinwhich is active in the specific cell or tissue types or by othertranscription factor, as compared to other cells or tissue types.Non-limiting specific examples of such an expression control element arehexokinase II, COX-2, α-fetoprotein, carcinoembryonic antigen, DE3/MUC1,prostate specific antigen, C-erB2/neu, glucose-dependent insulinsecretion stimulatory polypeptide (GIP), telomerase reversetranscriptase and a promoter such as hypoxia-responsive promoter.

According to the invention, a host cell transformed or transfected withIL-3Rα nucleic acid or vector of the invention is provided. Examples ofthe host cells, but are not limited to, include prokaryotic cell andeukaryotic cell, such as, bacteria, fungi (yeast), and cells of plants,insects and animals (e.g., mammals such as primates, human and thelike). Non-limiting examples of transformed cell include a bacteriatransformed with a recombinant bacteriophage nucleic acid, a plasmidnucleic acid or cosmid nucleic acid expression vector; a yeasttransformed with a recombinant yeast expression vector; a plant cellinfected with a recombinant virus expression vector (e.g., cauliflowermosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with arecombinant plasmid expression vector (e.g., Ti plasmid); an incest cellinfected with a recombinant virus expression vector (e.g., baculovirus);and an animal cell infected with a recombinant virus expression vector(e.g., retrovirus, adenovirus, vaccinia virus) or a transformed animalcell manipulated for stable expression. CHO cell is a non-limitingexample of a mammal host cell which expresses an IL-3Rα antibody and itssubsequence thereof and fragment. The host cell may be a plurality orpopulation of cells from a primary cell-separated line, an isolatedsecondary cell or subcultured cell, or an established cell line orimmortalized cell culture.

The term “be transformed” or be transfected” when it is used inreference to a cell (e.g., host cell) or an organism means a change ofgene in a cell after incorporation of an exogenous molecule, such as aprotein or a nucleic acid (e.g., transgene), into the cell. Accordingly,the “transfected” or “transformed” cell is a cell into which theexogenous molecule is introduced by the hand of man by, for example, byrecombinant DNA techniques or a progeny thereof.

The nucleic acid or protein can be transfected or transformed(expressed) in the cell or a progeny thereof stably or temporarily. Theintroduced protein can be expressed by growing the cell, or transcribingthe nucleic acid. Since there is a possibility that a mutation occursduring replication, there is a case that a progeny of the transfected ortransformed cell is not identical to the parent cell.

In general, a vector is used in the cell transfection or transformation.The vector can be included in a viral particle or vesicle and can beoptionally directed demands to a specific cell types by including aprotein on the particle or vesicle surface which binds to a target cellligand or receptor. Accordingly, a cell can be used as a target bypreparing the viral particle or vesicle itself or the viral surfaceprotein, for the purpose of an in vitro, ex vivo or in vivo transfectionor transformation. Accordingly, the vector includes in vitro, in vivoand ex vivo delivering techniques of viral and non-viral vectors into acell, tissue or organ.

In addition, introduction of a nucleic acid into a target cell (e.g., ahost cell) can also be carried out by a method conventionally known inthe technical field, such as osmotic shock (e.g., calcium phosphate),electroporation, microinjection, cell fusion and the like. Theintroduction of nucleic acid and polypeptide in vitro, ex vivo and invivo can also be carried out using other techniques. For example, apolymer substance such as polyester, poyamic acid, hydrogel, polyvinylpyrrolidone, ethylene-vinyl acetate, methyl cellulose,carboxymethylcellulose, protamine sulfate, or lactide/glycolidecopolymer, polylactide/glycolide copolymer, or ethylene vinyl acetatecopolymer and the like. The nucleic acid can be enclosed in ahydroxymethyl cellulose or gelatin-microcapsule, or a microcapsuleprepared using poly(methyl methacrylate microcapsule, or in a colloidsystem, respectively, by a coacervation technique or by interfacialpolymerization. The colloid dispersion system includes a system based ona polymer complex, nanocapsule, microsphere, beads and lipid(oil-in-water type emulsion, micelle, mixed micelle, liposome and thelike).

The liposome for introducing various compositions into cells isconventionally known in the technical field, and for example,phosphatidylcholine, phosphatidylserine, lipofectin and DOTAP areincluded therein (e.g., U.S. Pat. No. 4,844,904, U.S. Pat. No.5,000,959, U.S. Pat. No. 4,863,740 and U.S. Pat. No. 4,975,282; andGIBCO-BRL, Gaithersburg, Md.). Piperazine based amphilic cationic lipidswhich is useful in gene therapy (see e.g., U.S. Pat. No. 5,861,397) arealso known. A cationic lipid system is also known (see e.g., U.S. Pat.No. 5,459,127). In this specification, the polymer substance,microcapsule and colloid dispersion system (loposome and the like) arecollectively called as “vesicle”.

In addition, examples of the suitable techniques which can be used inthe method for producing an antibody are affinity purification,non-modified gel purification, HPLC or RP-HPLC, size exclusion,purification by protein A column and an optional combination of thesetechniques. An IL-3Rα antibody isotype can be determined using ELISAassay, and for example, human Ig can be identified using mouse Igabsorbed anti-human Ig.

Binding affinity can be determined by association (Ka) and dissociation(Kd) rates. The equilibrium affinity constant KD is the ratio of Ka/Kd.The association (Ka) and dissociation (Kd) rates can be measured usingsurface plasmon resonance (SPR) (Rich and Myszka, Curr. Opin.Biotechnol., 11: 54 (2000): Englebienne, Analyst., 123: 1599 (1998)).Instrumentation and methods for real time detection and monitoring ofassociation rate are conventionally known and commercially available(BiaCore 2000, Biacore AB, Upsala, Sweden; and Malmqvist, Biochem. Soc.Trans., 27:335 (1999)). The KD value can be defined as the IL-3Rαantibody concentration required to saturate one half of the binding site(50%) on IL-3Rα.

(Crossing Property in Primates)

Currently, although as many as 500 therapeutic antibodies are beingdeveloped in the world, it is said that human antibodies have a highpossibility to be able to avoid problems of immunogenicity. However, onthe other hand, there are many cases in which drug efficacy of humanantibodies are not exhibited at all in rodents. In that case, there aremany cases in that primates have to be used in toxicity tests, andfurthermore the reactivity is found only in chimpanzee is not rare inmany cases. When the pharmacological reaction can be found only inchimpanzee, the toxicity test is further significantly constrained. Inthe first place, facilities where chimpanzee experiments can be carriedout are considerably limited, individuals are infected with HIV in manycases and there are also problems of labor hygiene of workers involvedin the experiments. In addition, regarding chimpanzee, there are largelimitations that anatomy test after final drug administration cannot becarried out and of reproductive toxicity test is also impossible tocarry out and the like. Accordingly, the ability to verify drug efficacyin monkey (Macaca fascicularis and/or Macaca mulatta) is useful from theviewpoint of advancing toxicity tests which are essential for developingpharmaceuticals.

Regarding the method for confirming monkey crossreactivity with monkey,it can be confirmed by a conventionally known method such asimmunochemical tissue staining method, solid phase enzyme immunoassay(hereinafter, “ELISA”), flow cytometry (FCM) and the like.

(Pharmaceutical Composition)

Antibodies can be included in a pharmaceutical composition. In anembodiment, an antibody comprises a pharmaceutically acceptable carrier,a stabilizer or a filler and is prepared in the form of aqueous solutionor as a freeze-dried preparation. Typically, an appropriate amount of apharmaceutically acceptable salt is used for isotonicity of thepharmaceutical preparation. Examples of the acceptable carrier,stabilizer or filler include a buffer solution such as phosphate,citrate and other organic acid and the like; a low molecular weight(less than 10 in the number of residues) polypeptide; a protein such asserum albumin, gelatin, immunoglobulin and the like; a hydrophilicpolymer such as polyvinyl pyrrolidone; an amino acid such as glycine,glutamine, asparagine, histidine, arginine, lysine and the like; amonosaccharide, disaccharides and other carbohydrates such as glucose,mannose, dextrin and the like; a chelating agent such as EDTA and thelike; saccharides such as sucrose, mannitol, trehalose, sorbitol and thelike; a salt forming counter ion such as sodium and the like; a metalcomplex (e.g., Zn-protein complex); an antiseptic (octadecyldimethylbenzylammonium chloride; hexamethonium chloride; banzalconiumchloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkylparabens such as methyl or propyl paraben; catechol; resorcinol;cyclohexanol; 3-pentanol; and m-cresol); and/or a nonionic surfactantsuch as TWEEN™, PLURONICS™, polyethylene glycol (PEG) and the like.

(Therapeutic Use of Antitumor Substance which Targets IL-3Rα ExpressionCells)

Examples of the diseases for which the therapeutic use is examined, butare not limited thereto, include the diseases which can be considered totreat by binding or targeting IL-3Rα-expressing blood tumor cells (AMLcell, CML cell, MDS cell, ALL cell, CLL cell, multiple myeloma cell andthe like), mastocyte, basophile, helper T cell (e.g., Th1 cell, Th17cell), regulatory T cell (e.g., CD4 positive CD25 positive cell),antigen presenting cell (e.g., dendritic cell, monocyte.macrophage andrelated cells (hepatic stellate cell, osteoclast, microglia,intraepidermal macrophage, dust cell (alveolar phagocyte) and thelike)).

Examples of the disease for which therapeutic use is examined include ablood disease in which expression of IL-3Rα is found in bone marrow orperipheral blood. Specific example may include acute myeloid leukemia(AML). Based on the FAB classification (French-American-Britishcriteria) which can determine which stage of the cell among the cells inthe course of differentiating into various blood cells from thehematopoietic stem cell caused tumorigenic transformation, the acutemyeloid leukemia is classified into disease types of M0(micro-differentiation type myeloblastic leukemia), M1 (undifferentiatedmyeloblastic leukemia), M2 (differentiated myeloblastic leukemia), M3(acute promyelocytic leukemia), M4 (myelomonocytic leukemia), M5(monocytic leukemia), M6 (erythroleukemia), M7 (megakaryocytic leukemia)and subtypes thereof. In addition, further examples of diseases includeacute lymphocytic leukemia, atypical leukemia, chronic lymphocyticleukemia, adult T cell leukemia, NK/T cell lymphoma, granularlymphocytosis (LGL leukemia), polycythemia vera, essentialthrombocythemia, hypereosinophilic syndrome, Hodgkin lymphoma,non-Hodgkin lymphoma, follicular lymphoma, MALT lymphoma, mantle celllymphoma, diffuse large B-cell lymphoma, Burkitt lymphoma, lymphoblasticlymphoma and Catsleman disease.

The method of the invention which comprises administration or deliveryof an IL-3Rα antibody and an anti-tumor substance which targets anIL-3Rα expression cell can be carried out by any acceptable method. In aspecified embodiment, these are administered to a subject, locally,regionally or systemically.

In addition, regarding the IL-3Rα antibody, the antitumor substancewhich targets IL-3Rα expression cell for treating the above-mentioneddiseases can also be considered to combine with other therapeutic agentsuitable for the same disease (typically a chemotherapeutic agent) or beadministered in combination with radiotherapy. Examples of the suitableother therapeutic agent include a chemotherapeutic agent such ascytarabine (Ara-C), an anthracycline system antitumor agent (typically,daunorubicin (DNR), idarubicin (IDA)) and the like, a differentiationinduction therapeutic agent such as all-trans retinoic acid (ATRA),arsenious acid, Am80 (tamibarotene), gemtuzumab-ozogamicin (ozogamicinconjugate anti-CD33 antibody), topotecan, fludarabine, cyclosporine,mitoxantrone (MIT), interferon and imatinib, but are not limitedthereto, and also include a combination with a therapeutic methodconsidered to be clinically effective.

Mammals (e.g., human) are included in the subject which can be treatedby the invention. In a specified embodiment, it is a subject who is acandidate of blood tumor or a subject who received treatment of theblood tumor, a subject having a possibility causing IL-3Rα-mediatedcellular response or a subject who received treatment of theIL-3Rα-mediated cellular response, a subject who is a candidate of amyelocytic malignant tumor or a subject who received treatment of themyelocytic malignant tumor or a subject who is a candidate of acutemyeloid leukemia or a subject who received treatment of the acutemyeloid leukemia.

According to this specification, the terms “treat”, “treating”,“treatment” and the grammatical variations thereof mean a protocol, aplanning, a process or an improving method which is carried out on eachsubject who is desirable to obtain physiological effect or good outcomeon the patient. Accordingly, the method of the invention includes atreatment and a treating method which produce measurable improvement orbeneficial effect, particularly on a disorder, a disease, pathology, acondition of a disease or a symptom of a given subject. The measurableimprovement or profitable effect is objective or subjective, immoderate,transient or long-term improvement of any one of disorders, diseases,pathology, conditions of a disease or symptoms, or a reduction in onset,severity, duration or frequency of adverse symptom related to or causedby disorders, diseases, physiological conditions, pathology or state.According to the method of the invention, there is a possibility thatits effect is not always exhibited immediately, but eventual improvementor beneficial effect is found a little later with the lapse of time, sothat stabilization or amelioration in a give subject will occur.

Unless otherwise noted, all of the technical terms and scientific termsused in this specification have the same meanings of those which aregenerally evident for persons in the technical field to which theinvention is related. Methods and materials similar or equivalent tothose described in this specification can be used in the operations orexaminations of the invention, but those which are described in thisspecification are suitable methods and materials.

EXAMPLES Example 1 Preparation of Human, Macaca fascicularis or Macacamulatta IL-3Rα Expression Cell

(Molecular Cloning of IL-3Rα cDNA and Preparation of Expression Vector)

Human IL-3Rα cDNA was amplified from a blood cell-derived DNA (CLONTECHHuman MTC Panel) by PCR using ExTaq (TAKARA BIO INC.). As a PCR device,GeneAmp PCR System 9700 (Applied Biosystems, hereinafter, the PCR deviceis the same in this specification) was used. Regarding the PCR, after adenaturation step at 94° C. for 5 minutes, a three step reaction at 94°C. 30 seconds-55° C. 30 seconds-72° C. 2 minutes was carried out 40cycles and then a reaction at 99° C. for 30 seconds was carried out. ThePCR primers used are as follows.

IL-3Rα_Fw: (SEQ ID NO: 3) 5′-CGGCAATTGCCACCATGGTCCTCCTTTGGCTCAC-3′IL-3Rα_Re: (SEQ ID NO: 4) 5′-ATTGCGGCCGCTCAAGTTTTCTGCACGACCT-3′

The thus obtained PCR products were subjected to 0.8% agarose gelelectrophoresis (135 V, 15 minutes, TAE buffer). DNA was visualized byethidium bromide staining. A band at around 1.2 kb was cut out andextracted using JetSob (Genomed). The extracted DNA was digested withMfeI and NotI, mixed with pEGFP-N1 vector (Clontech) or pEF6/Myc-Hisvector which had been digested with EcoRI and NotI and ligated usingTaKaRa Ligation Kit. Regarding the transformation, the ligation sampleand a DH10B competent cell were mixed and spread on LB plate (containingkanamycin). Insert check of the pEGFP-N1 vector was carried out bycolony direct PCR using LA Taq (Takara Shuzo Co., Ltd.). Regarding thePCR, after a denaturation step at 94° C. for 5 minutes, a three stepreaction at 94° C. 30 seconds-55° C. 30 seconds-72° C. 2 minutes wascarried out 40 cycles and then a reaction at 99° C. for 30 seconds wascarried out. Regarding the primers used, IL-3Rα-Fw and IL-3Rα-Re wereused.

The thus obtained PCR products were subjected to 0.8% agarose gelelectrophoresis (135 V, 15 minutes, TAE buffer). DNA was visualized byethidium bromide staining. Using a colony from which amplification ataround 1.2 kb was obtained, nucleotide sequence was determined by adirect sequencing method. In the reaction of sequence samples, BigDye®Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) and GeneAmpPCR System 9700 (Applied Biosystems) were used (these were used in theall DNA sequence analyses in this specification). Regarding the primers,IL-3Rα-Fw, IL-3Rα-Re and the following primer were used.

(SEQ ID NO: 5) IL-3Rα_seqF1: 5′-GTCTTCACTACAAAACGGAT-3′

ABI 3700XL DNA analyzer (Applied Biosystems) was used as the sequenceanalyzing device. A clone having the same sequence of the coding regionof GenBank association number NP-002174.1 was selected and a plasmid DNAwas extracted by a Miniprep method. The vector names werepEGFR-N1/hCD123 and pEF6/Myc-His/hCD123, respectively.

The sequence of the insert (MfeI to NotI) was as follows.

(SEQ ID NO: 6) CAATTGCCACCATGGTCCTCCTTTGGCTCACGCTGCTCCTGATCGCCCTGCCCTGTCTCCTGCAAACGAAGGAAGATCCAAACCCACCAATCACGAACCTAAGGATGAAAGCAAAGGCTCAGCAGTTGACCTGGGACCTTAACAGAAATGTGACCGATATCGAGTGTGTTAAAGACGCCGACTATTCTATGCCGGCAGTGAACAATAGCTATTGCCAGTTTGGAGCAATTTCCTTATGTGAAGTGACCAACTACACCGTCCGAGTGGCCAACCCACCATTCTCCACGTGGATCCTCTTCCCTGAGAACAGTGGGAAGCCTTGGGCAGGTGCGGAGAATCTGACCTGCTGGATTCATGACGTGGATTTCTTGAGCTGCAGCTGGGCGGTAGGCCCGGGGGCCCCCGCGGACGTCCAGTACGACCTGTACTTGAACGTTGCCAACAGGCGTCAACAGTACGAGTGTCTTCACTACAAAACGGATGCTCAGGGAACACGTATCGGGTGTCGTTTCGATGACATCTCTCGACTCTCCAGCGGTTCTCAAAGTTCCCACATCCTGGTGCGGGGCAGGAGCGCAGCCTTCGGTATCCCCTGCACAGATAAGTTTGTCGTCTTTTCACAGATTGAGATATTAACTCCACCCAACATGACTGCAAAGTGTAATAAGACACATTCCTTTATGCACTGGAAAATGAGAAGTCATTTCAATCGCAAATTTCGCTATGAGCTTCAGATACAAAAGAGAATGCAGCCTGTAATCACAGAACAGGTCAGAGACAGAACCTCCTTCCAGCTACTCAATCCTGGAACGTACACAGTACAAATAAGAGCCCGGGAAAGAGTGTATGAATTCTTGAGCGCCTGGAGCACCCCCCAGCGCTTCGAGTGCGACCAGGAGGAGGGCGCAAACACACGTGCCTGGCGGACGTCGCTGCTGATCGCGCTGGGGACGCTGCTGGCCCTGGTCTGTGTCTTCGTGATCTGCAGAAGGTATCTGGTGATGCAGAGACTCTTTCCCCGCATCCCTCACATGAAAGACCCCATCGGTGACAGCTTCCAAAACGACAAGCTGGTGGTCTGGGAGGCGGGCAAAGCCGGCCTGGAGGAGTGTCTGGTGACTGAAGTACAGGTCGTGCAGAAAACTTGAGC GGCCGC

The Macaca fascicularis and Macaca mulatta cDNA samples were amplifiedfrom a Macaca fascicularis bone marrow-derived cDNA or Macaca mulattabone marrow-derived cDNA by a PCR method using LA Taq (TAKARA BIO INC).GeneAmp PCR System 9700 (Applied Biosystems) was used as the PCR device.Regarding the PCR, after a denaturation step at 95° C. for 1 minute, athree step reaction at 95° C. 15 seconds-56° C. 15 seconds-72° C. 70seconds was carried out 40 cycles and then a reaction at 72° C. for 2minutes was carried out. Subsequences were obtained through BLASTretrieval for the public data base of Macaca mulatta genome(http://www.hgsc.bnm.tmc.edu/blast.hgsc), based on the hIL-3Rα cDNAsequence to design primers. The used primer sequences were as follows.

Rhe123Fw1: (SEQ ID NO: 7) CGGCAATTGCCACCATGACCCTCCTTTGGCTGACGCTGRhe123Rv1: (SEQ ID NO: 8) TATATTGCGGCCGCTCAAGTTTTCTCCACCACCTGCAC

The thus obtained PCR products were subjected to 0.8% agarose gelelectrophoresis (135 V, 15 minutes, TAE buffer). DNA was visualized byethidium bromide staining. A band at around 1.2 kb was cut out and theDNA was extracted using Gel Extraction Kit (QIAGEN). The thus extractedDNA was mixed with pGEM-T Easy vector (Promega) and ligated using TaKaRaLigation Kit. Regarding the transformation, the ligation sample and aDH10B competent cell were mixed and spread on LB plate (containingampicillin). Insert check of the pGEM-T Easy vector was carried out bycolony direct PCR using LA Taq (Takara Shuzo Co., Ltd.). Regarding thePCR, after a denaturation step at 95° C. for 1 minute, a three stepreaction at 95° C. 15 seconds-56° C. 15 seconds-72° C. 1 minute wascarried out 35 cycles and then a reaction at 72° C. for 2 minutes wascarried out. The following were used as the primers.

T7: (SEQ ID NO: 9) TAATACGACTCACTATAGGG SP6: (SEQ ID NO: 10)GATTTAGGTGACACTATAG

The thus obtained PCR products were subjected to 0.8% agarose gelelectrophoresis (135 V, 15 minutes, TAE buffer). DNA was visualized byethidium bromide staining. Using a colony from which amplification ataround 1.2 kb was obtained, nucleotide sequence was determined by adirect sequencing method. As the PCR primers, T7 and SP6 were used. Aclone showing no mutation by PCR was selected and its plasmid DNA wasextracted by the Miniprep method. The thus obtained DNA was digestedwith MfeI and NotI, mixed with pEGFP-N1 vector (Clontech) which had beencleaved with EcoRI and NotI and ligated using TaKaRa Ligation Kit.Regarding the transformation, the ligation sample and a DH10B competentcell were mixed and spread on LB plate (containing kanamycin).

Insert check of the pEGFP-N1 vector was carried out by a colony directPCR using La Taq (Takara Shuzo Co., Ltd.). Regarding the PCR, after adenaturation step at 94° C. for 5 minutes, a three step reaction at 94°C. 30 seconds-55° C. 30 seconds-72° C. 2 minutes was carried out 40cycles and then a reaction at 99° C. for 30 seconds was carried out.Regarding the used PCR primers, Rhe123Fw1 and Rhe123Rv1 were used.

The thus obtained PCR products were subjected to 0.8% agarose gelelectrophoresis (135 V, 15 minutes, TAE buffer). DNA was visualized byethidium bromide staining. Using a colony from which amplification ataround 1.2 kb was obtained, nucleotide sequence was determined by adirect sequencing method. BigDye® Terminator v3.1 Cycle Sequencing Kit(Applied Biosystems) and GeneAmp PCR System 9700 (Applied Biosystems)were used in the reaction of sequence sample (these were used in all ofthe DNA sequence analyses in this specification). As the primers,Rhe123Fw1 and Rhe123Rv1 were used. The vectors were namedpEGFR-N1/cyCD123 and pEGFR-N1/rhCD123, respectively.

The sequence of the insert (MefI to NotI) of Macaca fascicularis IL-3Rαwas as follows.

(SEQ ID NO: 11) CAATTGCCACCATGACCCTCCTTTGGCTGACGCTGCTCCTGGTCGCCACGCCCTGTCTCCTGCAAACGAAGGAGGATCCAAATGCACCAATCAGGAATCTAAGGATGAAAGAAAAGGCTCAGCAGTTGATGTGGGACCTGAACAGAAACGTGACCGACGTGGAGTGTATCAAAGGCACCGACTATTCTATGCCGGCAATGAACAACAGCTATTGCCAGTTCGGAGCCATTTCCTTATGTGAAGTGACCAACTACACCGTCCGAGTGGCCAGTCCCCCGTTCTCCACGTGGATCCTCTTCCCTGAGAACAGTGGGACGCCTCAGGCAGGCGCGGAGAATCTGACCTGCTGGGTTCATGACGTGGATTTCTTGAGCTGCAGCTGGGTGGCAGGCCCGGCGGCCCCCGCTGACGTCCAGTACGACCTGTACTTGAACAATCCCAACAGCCACGAACAGTACAGGTGCCTTCACTACAAAACGGATGCTCGGGGAACACAGATCGGGTGTCGGTTCGATGACATCGCTCGACTCTCCCGCGGTTCTCAAAGTTCCCACATCCTGGTGAGGGGCAGGAGCGCAGCCGTCAGTATCCCCTGCACAGATAAGTTTGTCTTCTTTTCACAGATTGAGAGATTAACTCCACCCAACATGACTGGAGAGTGTAATGAGACACATTCCTTCATGCACTGGAAAATGAAAAGTCATTTCAATCGCAAATTCCGCTATGAGCTTCGGATCCAAAAGAGAATGCAGCCTGTAAGGACAGAACAGGTCAGAGACACAACCTCCTTCCAGCTACCCAATCCTGGAACGTACACAGTGCAAATAAGAGCCCGGGAAACAGTGTATGAATTCTTGAGTGCCTGGAGCACCCCCCAGCGCTTCGAGTGCGACCAGGAGGAGGGCGCGAGCTCGCGTGCCTGGCGGACGTCGCTGCTGATCGCGCTGGGGACGCTGCTGGCCTTGCTCTGTGTGTTCCTCATCTGCAGAAGGTATCTGGTGATGCAGAGGCTGTTTCCCCGCATCCCACACATGAAAGACCCCATCGGTGACACCTTCCAACAGGACAAGCTGGTGGTCTGGGAGGCGGGCAAAGCCGGCCTGGAGGAGTGTCTGGTGTCTGAAGTGCAGGTGGTGGAGAAAACTTGAGCGG CCGC

The sequence of the insert (MefI to NotI) of Macaca mulatta IL-3Rα wasas follows.

(SEQ ID NO: 12) CAATTGCCACCATGACCCTCCTTTGGCTGACGCTGCTCCTGGTCGCCACGCCCTGTCTCCTGCAAACCAAGGAGGATCCAAATGCACCAATCAGGAATCTAAGGATGAAAGAAAAGGCTCAGCAGTTGATGTGGGACCTGAACAGAAACGTGACCGACGTGGAGTGTATCAAAGGCACCGACTATTCTATGCCGGCAATGAACGACAGCTATTGCCAGTTCGGAGCCATTTCCTTATGTGAAGTGACCAACTACACCGTCCGAGTGGCCAGTCCTCCGTTCTCCACGTGGATCCTCTTCCCTGAGAACAGTGGGACGCCTCGGGCAGGCGCGGAGAATTTGACCTGCTGGGTTCATGACGTGGATTTCTTGAGCTGCAGCTGGGTGGTAGGCCCGGCGGCCCCCGCTGACGTCCAGTACGACCTGTACTTGAACAATCCCAACAGCCACGAACAGTACAGGTGCCTTCGCTACAAAACGGATGCTCGGGGAACACAGATCGGGTGTCGGTTCGATGACATCGCTCGACTCTCCCGCGGTTCTCAAAGTTCCCACATCCTGGTGAGGGGCAGGAGCGCAGCCGTCAGTATCCCCTGCACAGATAAGTTTGTCTTCTTTTCACAGATTGAGAGATTAACTCCACCCAACATGACTGGAGAGTGTAATGAGACACATTCCTTCATGCACTGGAAAATGAAAAGTCATTTCAATCGCAAATTCCACTATGAGCTTCGGATCCAAAAGAGAATGCAGCCTGTAAGGACAGAACAGGTCAGAGACACAACCTCCTTCCAGCTACCCAATCCTGGAACGTACACAGTGCAAATAAGAGCCCGGGAAACAGTGTATGAATTCTTGAGTGCCTGGAGCACCCCCCAGCGCTTCGAGTGCGACCAGGAGGAGGGCGCGAGCTCGCGTGCCTGGCGGACGTCGCTGCTGATCGCGCTGGGGACGCTGCTGGCCTTGCTCTGTGTGTTCCTCATCTGCAGAAGGTATCTGGTGATGCAGAGGCTGTTTCCCCGCATCCCACACATGAAAGACCCCATCGGTGACACCTTCCAACAGGACAAGCTGGTGGTCTGGGAGGCGGGCAAAGCCGGCCTGGAGGAGTGTCTGGTGTCTGAAGTGCAGGTGGTGGAGAAAACTTGAGCGG CCGC

(Preparation of IL-3Rα Forced Expression Cell Line)

L929 cell (manufactured by ATCC) and Colon-26 cell (manufactured byATCC) were infected with pEGFP-N1 vector/hCD123 or pEF6/Myc-Hisvector/hCD123 using electroporation (BTX). Specifically, 10 to 20 μg ofDNA was mixed with one hundred thousand cells and allowed to react at300 V and 950 μF. Regarding the cells, drug resistant cells wereselected using neomycin (Calbiochem) for pEGFP-N1/hCD123 or blasticidin(Invitrogen) for pEF6/Myc-His/hCD123. Regarding the thus selected cells,a GFP-positive cell or a cell highly expressing IL-3Rα (CD123) wasfurther selected by sorting using flow cytometry (FAC SVantage, FACSAriaand the like, BD Biosciences) and named as L929/hCD123 andColon-26/hCD123, respectively.

Regarding the preparation of Macaca fascicularis IL-3Rα and Macacamulatta IL-3Rα forced expression cells, these were also prepared usingL929 and Colon-26 in the same manner as the case of human IL-3Rα forcedexpression cell and named L929/cyCD123, Colon-26/cyCD123, L929/rhCD123and Colon-26/rhCD123.

Example 2 Preparation of Soluble Form of IL-3Rα Extracellular Region(Preparation of Soluble Form of Human IL-3Rα Extracellular RegionExpression Vector)

A cDNA encoding the extracellular region of human IL-3Rα was amplifiedby a PCR method, and FLAG tag was connected to its downstream.Specifically, the cDNA encoding the extracellular region of human IL-3Rαwas amplified by PCR using pEF6/Myc-His/hCD123 plasmid DNA as thetemplate and using Platinum Pfu polymerase (Invitrogen). Regarding thePCR, after a denaturation step at 96° C. for 2 minutes, a three stepreaction at 96° C. 20 seconds-55° C. 30 seconds-68° C. 65 seconds wascarried out 30 cycles. The primers used were IL-3Rα-Fw and the followingprimer.

hIL-3Rαsol-FLAG-NotI: (SEQ ID NO: 13)5′-ATTGCGGCCGCTCACTTATCGTCGTCATCCTTGTAGTCCCGCCAG GCACGTGTGTTTG-3′

The thus obtained PCR products were subjected to 0.8% agarose gelelectrophoresis (135 V, 15 minutes, TAE buffer). DNA was visualized byethidium bromide staining. The DNA was extracted using JetSorb(Genomed). Thus purified DNA was digested with MfeI and NotI and againsubjected to 0.8% agarose gel electrophoresis (135 V, 15 minutes, TAEbuffer). A band of around 1.0 kb was cut out and the DNA was extractedusing JetSorb (Genomed). The obtained DNA was mixed with apTracer-CMV/Bsd vector, which had been cleaved using the same enzymes ofthe purified DNA, and ligated using TaKaRa Ligation Kit. Regarding thetransformation, the ligation sample and a DH10B competent cell weremixed and spread on LB plate (containing ampicillin). Insert check wascarried out by colony direct PCR using LA Taq (Takara Shuzo Co., Ltd.).Regarding the PCR, after a denaturation step at 95° C. for 1 minute, athree step reaction at 95° C. 15 seconds-56° C. 15 seconds-72° C. 40seconds was carried out 35 cycles and then an elongation reaction at 72°C. for 2 minutes was carried out. The PCR primers used were IL-3Rα-Fwand IL-3Rαsol-FLAG-NotI.

The thus obtained PCR products were subjected to 0.8% agarose gelelectrophoresis (135 V, 15 minutes, TAE buffer). DNA was visualized byethidium bromide staining. A plasmid DNA was extracted by the Miniprepmethod from a colony in which amplification of around 1.0 kb wasobtained. It was found by a DNA sequence analysis that the purifiedplasmid DNA has the sequence identical to the corresponding region ofGenBank accession number NP-002174.1.

The sequence of the insert (MfeI to NotI) was as follows.

(SEQ ID NO: 14) CAATTGCCACCATGGTCCTCCTTTGGCTCACGCTGCTCCTGATCGCCCTGCCCTGTCTCCTGCAAACGAAGGAAGATCCAAACCCACCAATCACGAACCTAAGGATGAAAGCAAAGGCTCAGCAGTTGACCTGGGACCTTAACAGAAATGTGACCGATATCGAGTGTGTTAAAGACGCCGACTATTCTATGCCGGCAGTGAACAATAGCTATTGCCAGTTTGGAGCAATTTCCTTATGTGAAGTGACCAACTACACCGTCCGAGTGGCCAACCCACCATTCTCCACGTGGATCCTCTTCCCTGAGAACAGTGGGAAGCCTTGGGCAGGTGCGGAGAATCTGACCTGCTGGATTCATGACGTGGATTTCTTGAGCTGCAGCTGGGCGGTAGGCCCGGGGGCCCCCGCGGACGTCCAGTACGACCTGTACTTGAACGTTGCCAACAGGCGTCAACAGTACGAGTGTCTTCACTACAAAACGGATGCTCAGGGAACACGTATCGGGTGTCGTTTCGATGACATCTCTCGACTCTCCAGCGGTTCTCAAAGTTCCCACATCCTGGTGCGGGGCAGGAGCGCAGCCTTCGGTATCCCCTGCACAGATAAGTTTGTCGTCTTTTCACAGATTGAGATATTAACTCCACCCAACATGACTGCAAAGTGTAATAAGACACATTCCTTTATGCACTGGAAAATGAGAAGTCATTTCAATCGCAAATTTCGCTATGAGCTTCAGATACAAAAGAGAATGCAGCCTGTAATCACAGAACAGGTCAGAGACAGAACCTCCTTCCAGCTACTCAATCCTGGAACGTACACAGTACAAATAAGAGCCCGGGAAAGAGTGTATGAATTCTTGAGCGCCTGGAGCACCCCCCAGCGCTTCGAGTGCGACCAGGAGGAGGGCGCAAACACACGTGCCTGGCGGGACTACAAGGATGACGACGATAAGTGAGCGGCCG C

(Preparation of Soluble Form of Human IL-3Rα Protein)

Plasmid DNA of pTracer CMV expression vector containing soluble form ofIL-3Rα sequence was purified using QIAGEN Plasmid Maxi Kit. A CHOras1cell was used as a host cell for expression. The CHOras1 cell wascultured with shaking using SFM II medium (Invitrogen) (37° C., 5% CO₂).

A PEI method was used in the gene introduction. Polyethylenimine,Linear, MW 25,000 (Polysciences) was weighed and dissolved in PBS whileadjusting to around pH 7.0 with HCl (1 g/l). The obtained solution wasstirred for 1 hour and then sterilized by filtering through a membranefilter having a pore size of 0.22 μm, MILLEX-GV (Millipore). Then, 1 mgof the purified plasmid DNA was mixed with 20 ml of Opti-Pro SFM(Invitrogen) to obtain Solution A. Solution B was prepared by mixing 2.5ml of PEI solution (1 g/l) with 20 ml of Opti-Pro SFM (Invitrogen).After solution A and Solution B were mixed, and then allowed to standstill for 10 minutes, the obtained solution was added to CHOras1 cells(1,000,000 cells per 1 ml). After six days, the cell supernatant wasrecovered and used for the protein purification.

Purification of the soluble form of human IL-3Rα protein was carried outby the following method. A culture supernatant containing soluble formof IL-3Rα protein was recovered by centrifugation 6 days after the geneintroduction and passed through a filter. The obtained solution wasdiluted 5 times with Tris buffered saline (TBS), an Anti-FLAG column wasprepared using anti-FLAG M2 Agarose Affinity Gel (Sigma) and thesolution was applied thereto using HiLoad Pump P-50 (Pharmacia Biotech).Elution was carried out using FLAG peptide (Sigma) and in accordancewith the manual. The eluate was fractioned into several fractions, eachfraction was subjected to SDS-PAGE (MultiGel II Mini 10/20% gradientgel; Cosmo Bio Co., Ltd.) under a reducing condition, and then silverstaining and Western blotting were carried out. A silver stainingreagent “Daiichi” (Daiichi Pure Chemicals Co., Ltd.) was used for thesilver staining. Anti-FLAG M2 antibody (Sigma) and an alkalinephosphatase-labeled rabbit anti-mouse immunoglobulin antibody were usedfor the Western blotting. A fraction in which the protein of interestwas found was concentrated using Amicon Ultra-4 10K (Millipore), and gelfiltration chromatography was carried out using Superdex 200 gp (GEHealthcare). After fractionation, each fraction was subjected toSDS-PAGE (MultiGel II Mini 10/20% gradient gel; Cosmo Bio Co., Ltd.)under a reducing condition, and then silver staining and Westernblotting were carried out. A silver staining reagent “Daiichi” (DaiichiPure Chemicals Co., Ltd.) was used in the silver staining. Anti-FLAG M2antibody (Sigma) and an alkaline phosphatase-labeled rabbit anti-mouseimmunoglobulin antibody were used in the Western blotting. A fraction inwhich the protein of interest was found was concentrated using AmiconUltra-4 10K (Millipore) and washed with PBS. By carrying outsterilization by filtration using a membrane filter MILLEX-GV(Millipore) having a pore size of 0.22 μm, a soluble form of humanIL-3Rα protein was obtained. As a result of Limulus test using LimulusES-II Kit Wako (Wako Pure Chemical Industries, Ltd.), endotoxin was notdetected. Regarding concentration of the soluble form of human IL-3Rαprotein, absorbance at 280 nm was measured and 1 mg/ml was calculated as1.4 OD.

Example 3 Preparation of Anti-Human IL-3Rα Human Antibody Using HumanAntibody Producing Mouse (Human Antibody Producing Mouse)

The mouse used in the immunization has a genetic background ofhomozygote on both of endogenous Ig heavy chain and κ light chaindisruptions and also simultaneously keeps the 14^(th) chromosomalfragment containing human Ig heavy chain locus (SC20) and human Ig κchain transgene (KCo5). This mouse was prepared by the crossing of aline A mouse having the human Ig heavy chain locus onto a line B mousehaving the human Ig κ chain transgene. The line A is a homozygote onboth of endogenous Ig heavy chain and κ light chain disruptions, is amouse line which maintain the 14^(th) chromosomal fragment (SC20) whichcan be transmitted to progeny and is described for example in a reportby Tomizuka et al. [Tomizuka et al., Proc. Natl. Acad. Sci. USA, 2000,Vol. 97: 722]. Also, the line B is a homozygote on both of endogenous Igheavy chain and κ light chain disruptions, is a mouse line (transgenicmouse) which maintains a human Ig κ chain transgene (KCo5) and isdescribed in a report by such as Fishwild et al. [Nat. Biotechnol(1996), 114: 845].

An individual in which human Ig heavy chain and κ light chain weresimultaneously detected in serum, obtained by the crossing of a line Amale mouse onto a line B female mouse or the crossing of a line A femalemouse onto a line B male mouse, [Ishida & Lonberg, IBC's 11^(th)Antibody Engineering, Abstract 2000] was used in the following immunetest. In this connection, the aforementioned human antibody producingmouse (referred to as KM mouse) can be obtained from Kyowa Hakko KirinCo., Ltd. by establishing a contract.

(Preparation of Human Monoclonal Antibody to Human IL-3Rα)

Regarding the preparation of monoclonal antibody in this Example, it wasprepared in accordance with a general method described in A Guide toMonoclonal Antibody Experimental Operations (written in Japanese)(edited by Tamie Ando et al. published by Kodansha, 1991) and the like.For the IL-3Rα as an immunogen, an IL-3Rα expressing L929 cell (CCL-1,ATCC), an IL-3Rα expressing Colon-26 cell (Cell Resource Center forBiomedical Research Institute of Development, Aging and Cancer TohokuUniversity) or a soluble form of human IL-3Rα Fc fusion protein wasused. As an animal to be immunized, the above-mentioned KM mouse wasused.

For the purpose of preparing human monoclonal antibody to human IL-3Rα,the KM mouse was immunized with the IL-3Rα expression L929 cell orIL-3Rα expression Colon-26 cell prepared in Example 1, intraperitoneallyat a dose of 1×10⁷ cells/animal every 1 week to 2 weeks in a total of 4times. Three days before the extraction of spleen which is describedbelow, 20 μg/mouse individual of the soluble form of human IL-3Rαprotein was administered through the caudal vein.

After the spleen was surgically obtained from the immunized mouse, thespleen was put into PBS and minced on a mesh (cell strainer, FALCON)using a syringe piston. After the cell suspension was passed through themesh and was centrifuged, the obtained precipitated cells werere-suspended in Red Blood Cell Lysing Buffer (Sigma). After 5 minutes ofincubation at room temperature, serum-free DMEM medium (Invitrogen)containing 350 mg/ml sodium bicarbonate, 50 units/ml penicillin and 50μg/ml streptomycin (hereinafter referred to as “serum-free DMEM medium”)was added thereto to precipitate the cells. By suspending again in theserum-free DMEM medium, the number of cells was measured.

On the other hand, a myeloma cell SP2/0 (ATCC No. CRL-1581) was culturedat 37° C. in the presence of 5% carbon dioxide using DMEM medium(Invitrogen) containing 10% FCS (Invitrogen), 50 units/ml penicillin and50 μg/ml streptomycin (hereinafter referred to as “serum-containing DMEMmedium”). The SP2/0 cells were washed with serum-free DMEM medium. Inthe same manner, the cells were suspended in serum-free DMEM medium tomeasure the number of cells. After the suspension of the recoveredspleen-derived cells and a suspension of the mouse myeloma were mixed ata cell number ratio of 5:1, the mixed suspension was centrifuged, andthen the supernatant was completely removed. As a fusion agent, 50%(w/v) polyethylene glycol 1500 (Boehringer-Mannheim) was slowly added tothe obtained pellet while stirring the pellet with the tip of a pipette,and then serum-free DMEM medium heated to 37° C. in advance was slowlyadded thereto. Furthermore, an appropriate amount of serum-free DMEMmedium was slowly added thereto. Thereafter, the obtained solution wasallowed to stand still at 37° C. for 5 minutes in the presence of 5%carbon dioxide. After centrifugation, the supernatant was removed andthus obtained fused cells were suspended in DMEM medium (Invitrogen)containing 10% FCS (Invitrogen), penicillin-streptomycin-glutamine(Sigma), IL-6 (5 ng/ml) and 2-mercaptoethanol (Invitrogen) (hereinafter,referred to as “IL-6-containing DMEM medium”) and cultured at 37° C. inthe presence of 5% carbon dioxide. On the next day, the cells wererecovered by pipetting, and precipitated by centrifugation. The obtainedcell pellet was re-suspended in the IL-6-containing DMEM medium. Thesuspended cells were subjected to limiting dilution on a 96-well plateand cultured for about 7 days to 14 days. The culture supernatant wasused in the hybridoma screening described in the following example.

(Screening of Hybridoma Producing a Human Monoclonal Antibody whichBinds to Human IL-3Rα)

Screening of hybridoma was carried out using the cell supernatantprepared in the above example. The method was, in short, carried out bya flow cytometry in which a human IL-3Rα stable expression cell line wasused.

Specifically, a combination of human IL-3Rα expression L929 cell andparent cell line L929 cell or a combination of human IL-3Rα expressionColon-26 cell and parent cell line Colon-26 cell, was mixed with thesupernatant of hybridoma and allowed to stand still at 4° C. for 30minutes. After washing the obtained cells twice with a staining medium(Dulbecco's PBS containing 2% fetal calf serum, 2 mM EDTA, 0.05% NaN₃),Goat F(ab′)₂ Anti-Human IgG-PE (Southern Biotech) as a secondaryantibody was added thereto and allowed to stand still at 4° C. for 30minutes. After washing twice with the staining medium, the obtainedcells were analyzed by FACSCalibur (BD Biosciences). A hybridoma whichreacted with only human IL-3Rα expression L929 cell was collected.

The selected hybridoma was subjected to limiting dilution, and screeningwas carried out using its culture supernatant. Specifically, each ofhuman IL-3Rα expression L929 cell and parent cell line L929 cell wasmixed with the supernatant of hybridoma and allowed to stand still at 4°C. for 30 minutes. After washing twice with the staining medium, GoatF(ab′)₂ Anti-Human kappa-PE (Dako) as a secondary antibody was addedthereto and allowed to stand still at 4° C. for 30 minutes. Afterwashing twice with the staining medium, the cells were analyzed byFACSCalibur (BD Biosciences). A hybridoma which reacted with only humanIL-3Rα expression L929 cell was collected.

Example 4 Preparation of Recombinant Anti-Human IL-3Rα Human Antibody

(Obtaining of Anti-Human IL-3Rα Human Antibody Gene from Hybridoma andPreparation of Expression Vector)

From the hybridoma obtained in Example 3, clone names Old 4, Old5,Old17, Old19, New102 and Old6 were cultured using eRDF medium (KyokutoPharmaceutical Industrial Co., Ltd.) containing 10 ng/ml IL-6 (R & DSystems) and 10% Fetal Bovine Serum (SIGMA) and then cells werecollected by centrifugation. To the obtained cells, and then TRIZOL(GIBCO) was added and total RNA was extracted in accordance with theinstructions. Cloning of variable region of the antibody cDNA wascarried out using SMART RACE cDNA Amplification Kit (Clontech) inaccordance with the instructions attached thereto.

In addition, variable region was cloned from a hybridoma ATCC, HB12009,which produces an anti-human IL-3Rα mouse antibody 7G3 and was used as acontrol. By connecting the thus obtained cDNA with a DNA encoding humanIgG1 constant region, a chimeric antibody expression vector wasprepared. Specifically, the cells were collected by centrifugation fromthe cryopreserved hybridoma, and then TRIZOL (GIBCO) was added theretoto extract total RNA in accordance with the instructions. Cloning ofvariable region of the antibody cDNA was carried out using mouse IgGantibody-specific primes in addition to the SMART RACE cDNAAmplification Kit (Clontech), in accordance with the instructionsattached thereto.

Using 5 μg of the total RNA as a template, the 1st strand cDNA wasprepared.

1) Synthesis of the 1st Strand cDNA

Total RNA 5 μgm/3 μL,

5′CDS 1 μL

SMART oligo 1 μL

After the reaction mixture comprising the above compositions wasincubated at 72° C. for 2 minutes,

5× Buffer 2 μL

DTT 1 μL

DNTP mix 1 μL and

SuperscriptII 1 μL

were added to the reaction mixture and incubated at 42° C. for 1.5hours.

To the obtained mixture, 100 μl of Tricine Buffer was further added andincubated at 72° C. for 7 minutes.

2) Amplification of Heavy Chain Gene and Light Chain Gene by PCR andConstruction of Expression Vector of Recombinant Antibody

For amplification of cDNA, Z-Taq manufactured by Takara was used.

cDNA 2 μL

10×Z-Taq Buffer 5 μL

dNTPmix 4 μL

Z-Taq 1 μL

Primer 1

Primer 2

A reaction solution comprising the above-mentioned composition wasadjusted to a final volume of 50 μl with re-distilled water to besubjected to PCR.

For amplification of the heavy chain, UPM (SMART RACE cDNA AmplificationKit; manufactured by Clontech) and hh-6 primer

(SEQ ID NO: 15) (5′-GGTCCGGGAGATCATGAGGGTGTCCTT-3′)were used and a cycle of reactions at 98° C. 1 second and 68° C. 30seconds was repeated 30 times. Further, using 1 μl of the obtainedreaction solution as a template and using NUP (SMART RACE cDNAAmplification Kit; manufactured by Clontech) and hh-3 primer

(SEQ ID NO: 16) (5′-GTGCACGCCGCT GGT CAGGGCGCCTG-3′)as primers, a cycle of reactions at 98° C. 1 second and 68° C. 30seconds was repeated 20 times. Thereafter, the amplified PCR productswere purified using PCR purification kit (QIAGEN), and nucleotidesequences was determined using hh-4

(SEQ ID NO: 17) (5′-GGT GCC AGG GGG AAG ACC GAT GG-3′)as a primer. The following specific primers were synthesized based onsequence information, and the sequences were also determined from theopposite direction using the following primers.

Old4 heavy chain specific primer Fw (SEQ ID NO: 18)(5′-AGAGAGAGAGGTCGACCACCATGGACTGGACCTGGAGGTTCCT CTTTG T-3′)Old4 heavy chain specific primer Rv (SEQ ID NO: 19)(5′-AGAGAGAGAGGCTAGCTGAAGAGACGGTGACCATTGTCCC-3′)Old5 heavy chain specific primer Fw (SEQ ID NO: 20)(5′-AGAGAGAGAGGTCGACCACCATGGACTGGACCTGGAGGTTCCT CT TTG T-3′)Old5 heavy chain specific primer Rv (SEQ ID NO: 21)(5′-AGAGAGAGAGGCTAGCTGAAGAGACGGTGACCATTGTCCC-3′)O1d17 heavy chain specific primer Fw (SEQ ID NO: 22)(5′-AGAGAGAGAGGTCGACCACCATGGACTGGACCTGGAGGTTCCT CT TTG T-3′)O1d17 heavy chain specific primer Rv (SEQ ID NO: 23)(5′-AGAGAGAGAGGCTAGCTGAGGAGACGGTGACAAGGGTTCCC-3′)Old19 heavy chain specific primer Fw (SEQ ID NO: 24)(5′-AGAGAGAGAGGTCGACCACCATGGACTGGACCTGGAGGTTCCTC T TTG T-3′)Old19 heavy chain specific primer Rv (SEQ ID NO: 25)(5′-AGAGAGAGAGGCTAGCTGAGGAGACGGTGACCAGGGTTC-3′)New102 heavy chain specific primer Fw (SEQ ID NO: 26)(5′-AGAGAGAGAGGTCGACCACCATGGACTGGACCTGGAGGTTCCT CTTTG T-3′)New102 heavy chain specific primer Rv (SEQ ID NO: 27)(5′-AGAGAGAGAGGCTAGCTGAGGAGACGGTGACCAGGGTT-3′)Old6 heavy chain specific primer Fw (SEQ ID NO: 28)(5′-AGAGAGAGAGGTCGACCCACCATGGAACTGGGGCTCCGCTG-3′)Old6 heavy chain specific primer Rv (SEQ ID NO: 29)(5′-AGAGAGAGAGGCTAGCTGAGGAGACGGTGACCAGGGTTC-3′)

For the amplification of the heavy chain of mouse antibody 7G3, UPM(SMART RACE cDNA amplification Kit; manufactured by Clontech) and

mH-Rv1 primer (SEQ ID NO: 30)(5′-ATTTTG TCG ACC KYG GTS YTG CTG GCY GGGTG-3′) were used and a cycle of reactions at 98° C. 1 second and 68° C. 30seconds was repeated 30 times. Further, using 1 μl of this reactionsolution as a template and using NUP (SMART RACE cDNA Amplification Kit;manufactured by Clontech) and mH-

Rv2 primer (SEQ ID NO: 31) (5′-GCACACYRCTGGACAGGGATCCAGAGTTCC-3′),a cycle of reactions at 98° C. 1 second and 68° C. 30 seconds wasrepeated 20 times. Thereafter, the amplified PCR products were purifiedusing PCR purification kit (QIAGEN), and the nucleotide sequence ofheavy chain variable region was determined by using mH-Rv2 primer (SEQID NO:31) as a primer. The following specific primers were synthesizedbased on sequence information, and the sequences were also determinedfrom the opposite direction using the following primers.

7G3 heavy chain specific primer Fw (SEQ ID NO: 32)(5′-AGAGAGAGAGGTCGACCACCATGGGATGGAGCTGGATCTT TCTC-3′)7G3 heavy chain specific primer Rv (SEQ ID NO: 33)(5′-AGAGAGAGAGGCTAGCTGCAGAGACAGTGACCAGAGTCCC-3′)

PCR was carried out using the above-mentioned specific primers (98° C. 1second, 60° C. 30 seconds, 72° C. 30 seconds), and heavy chainamplification cDNA fragment was digested with SalI and NheI and insertedinto a N5KG1-Val Lark vector [a modified vector of N5KG1 (U.S. Pat. No.6,001,358, Idec Pharmaceuticals)] which had been cleaved with the sameenzymes. By determining the sequence using the vector as a template, itwas found that the inserted sequence is identical to the one determinedby direct sequence.

The light chain was amplified using UPM (SMART RACE cDNA AmplificationKit; manufactured by Clontech) and

hk-2 primer (SEQ ID NO: 34) (5′-GTT GAAGCT CTT TGT GAC GGG CGA GC-3′)and repeating a cycle of reactions at 98° C. 1 second and 68° C. 30seconds 30 times. Further, using 1 μl of this reaction solution as atemplate and using NUP (SMART RACE cDNA Amplification Kit; manufacturedby Clontech) and

hk-6  (SEQ ID NO: 35) (5′-TGGCGGGAAGATG AAG ACA GAT GGT G-3′),a cycle of reactions at 98° C. 1 second and 68° C. 30 seconds wasrepeated 20 times. Thereafter, the amplified PCR products were purifiedusing PCR purification kit (QIAGEN), and the nucleotide sequence wasdetermined using hk-6 primer. The following specific primers weresynthesized based on sequence information, and the sequences weredetermined also from the opposite direction.

Old4 light chain specific primer Fw (SEQ ID NO: 36)(5′-AGAGAGAGAGATCTCTCACCATGGACATGAGGGTCC CCG  CTC AGC-3′)Old4 light chain specific primer Rv (SEQ ID NO: 37)(5′-AGAGAGAGAGCGTACGTTTGATCTCCAGCTTGGTCC CCT G-3′)Old5 light chain specific primer Fw (SEQ ID NO: 38)(5′-AGA GAGAGAGATCTCTCACCATGGACATGAGGGTCCCC G CTC AGC-3′)Old5 light chain specific primer Rv (SEQ ID NO: 39)(5′-AGAGAGAGAGCGTACGTTTGATCTCCAGCTTGGTCC CCT G-3′)Old17 light chain specific primer Fw (SEQ ID NO: 40)(5′-AGAGAGAGAGATCTCTCACCATGGACATGAGGGTCC TCG CTC AG-3′)Old17 light chain specific primer Rv (SEQ ID NO: 41)(5′-AGAGAGAGAGCGTACGTTTGATCTCCAGCTTGGTCC CCT G-3′)Old19 light chain specific primer Fw (SEQ ID NO: 42)(5′-AGAGAGAGAGATCTCTCACCATGGACATGAGGGTCC TCG CTC AG-3′)Old19 light chain specific primer Rv (SEQ ID NO: 43)(5′-AGAGAGAGAGCGTACGTTTGATTTCCACCTTGGTCC CTT GGC-3′)New102 light chain specific primer Fw (SEQ ID NO: 44)(5′-AGAGAGAGAGATCTCTCACCATGGACATGAGGGTCC TCG CTC AG-3′)New102 light chain specific primer Rv (SEQ ID NO: 45)(5′-AGAGAGAGAGCGTACGTTTGATCTCCAGCTTGG TCC CCT G-3′)Old6 light chain specific primer Fw (SEQ ID NO: 46)(5′-AGAGAGAGAGATCTCTCACCATGGACATGAGGGTCCCCGC TCAGC-3′)Old6 light chain specific primer Rv (SEQ ID NO: 47)(5′-AGAGAGAGAGCGTACGTTTGATATCCACTTTGGTCCCAGGGC-3′)

Light chain of the mouse antibody 7G3 was amplified using UPM (SMARTRACE cDNA amplification Kit; manufactured by Clontech) and

mK-Rv1 primer mK_Rv1 (SEQ ID NO: 48)(5′-TT GAA GCT CTT GAC AAT GGG TGA AGT TGAT-3′)and repeating a cycle of reactions at 98° C. 1 second and 68° C. 30seconds 30 times. Further, using 1 μl of this reaction solution as atemplate and using NUP (SMART RACE cDNA Amplification Kit; manufacturedby Clontech) and

mK-Rv2 (SEQ ID NO: 49) (5′-GTAGGTGCTGTCTTTGCTGTCCTGATCAGT-3′),a cycle of reactions at 98° C. 1 second and 68° C. 30 seconds wasrepeated 20 times. Thereafter, the amplified PCR products were purifiedusing PCR purification kit (QIAGEN), and the nucleotide sequence wasdetermined using mK-Rv2 primer. The following specific primers weresynthesized based on sequence information, and the sequences were alsodetermined from the opposite direction.

7G3 light chain specific primer Fw (SEQ ID NO: 50)(5′-AGAGAGAGAGAGATCTCACCATGGAATCACAGACTCAGGTCC TC-3′)7G3 light chain specific primer Rv (SEQ ID NO: 51)(5′-AGAGAGAGAGCGTACGTTTTATTTCCAGCTTGGTCCCCCC-3′)

PCR was carried out using the above-mentioned specific primers (98° C. 1second, 60° C. 30 seconds, 72° C. 30 seconds), and s light chainamplification cDNA fragment was digested with BglII and BsiWI andinserted into a N5KG1-Val Lark vector which had been cleaved with thesame enzymes. By determining the sequence using the vector as atemplate, it was found that the inserted sequence is identical to theone determined by direct sequence.

Each of DNA molecules encoding the heavy chain variable region and lightchain variable region of Old4 and amino acid sequences of the heavychain variable region and light chain variable region are shown in thefollowing.

<Old4 heavy chain variable region> (SEQ ID NO: 52)GACCCGTCGACCACCATGGACTGGACCTGGAGGTTCCTCTTTGTGGTGGCAGCAGCTACAGGTGTCCAGTCCCAGGTCCAGCTGCTACAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCATGCAAGGCTTCTGGAGGCACCTTCAGCACCTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGTATAGTAAACTACGCACAGAAGTTCCAGGGCAGAGTCACGATTACCGCGGACGAATCCACGAGTACAGCCTACATGGAACTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTATTGTGCGAGAGGGGGGGGCTCGGGCCCAGATGTTCTTGATATCTGGGGCCAAGGGACAATGGTCACC GTCTCTTCAGCTAGCACCAA<Old4 heavy chain variable region> (SEQ ID NO: 53)MDWTWRFLFVVAAATGVQSQVQLLQSGAEVKKPGSSVKVSCKASGGTFSTYAISWVRQAPGQGLEWMGGIIPIFGIVNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGGGSGPDVLDIWGQGTMVTVSSAS TX

The translation initiation site of the heavy chain DNA is an ATG codonwhich starts from adenine (A) at position 16 from the 5′-terminal of SEQID NO:52, and a boundary between the antibody variable region and theconstant region is located between adenine (A) at position 432 andguanine (G) at position 433 from the 5′-terminal. In the heavy chainamino acid sequence, the heavy chain variable region is up to serine (S)residue at position 139 from the N-terminal of SEQ ID NO:53, and theconstant region is on and after alanine (A) at position 140. By a genesequence estimation software (Signal P ver. 2), the signal sequence ofheavy chain was estimated to be up to serine (S) at position 19 from theN-terminal of SEQ ID NO:53. The N-terminal of the mature form wasconsidered to be glutamine (Q) at position 20 of SEQ ID NO:53.

<Old4 light chain variable region> (SEQ ID NO: 54)CACAGATCTCTCACCATGGACATGAGGGTCCCCGCTCAGCTCCTGGGGCTCCTGCTGCTCTGGCTCCCAGGTGCCAGATGTGTCATCTGGATGACCCAGTCTCCATCCTTACTCTCTGCATCTACAGGAGACAGAGTCACCATCAGTTGTCGGATGAGTCAGGGCATTAGGAGTTATTTAGCCTGGTATCAGCAAAAACCAGGGAAAGCCCCTGAGCTCCTGATCTATGCTGCATCCACTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGTCTGAAGATTTTGCAACTTATTACTGTCAACAGTATTATAGTTTCCCGTACACTTTTGGCCAGGGGACCAAGCTGGAGATCAAACGTACGGTGG <Old4 light chain variable region>(SEQ ID NO: 55) MDMRVPAQLLGLLLLWLPGARCVIVVMTQSPSLLSASTGDRVTISCRMSQGIRSYLAWYQQKPGKAPELLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQSEDFATYYCQQYYSFPYTFGQGTKLEIKRTVX

The translation initiation site of the light chain DNA is an ATG codonwhich starts from adenine (A) at position 16 from the 5′-terminal of SEQID NO:54, and a boundary between the variable region and the constantregion of the antibody is located between adenine (A) at position 402and cytosine (C) at position 403 from the 5′-terminal. In the lightchain amino acid sequence, the light chain variable region is up tolysine (K) residue at position 129 from the N-terminal of SEQ ID NO:55,and the constant region is on and after arginine (R) at position 130. Bya gene sequence estimation software (Signal P ver. 2), the signalsequence of light chain was estimated to be up to cysteine (C) atposition 22 from the N-terminal of SEQ ID NO:55. The N-terminal of themature form was considered to be valine (V) at position 23 of SEQ IDNO:55.

Each of DNA molecules encoding the heavy chain variable region and thelight chain variable region of Old5 and amino acid sequences of theheavy chain variable region and light chain variable region was shown inthe following.

<Old5 heavy chain variable region> (SEQ ID NO: 56)GTCGACCACCATGGACTGGACCTGGAGGTTCCTCTTTGTGGTGGCAGCAGCTACAGGTGTCCAGTCCCAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCATGCAAGGCTTCTGGAGGCACCTTCAGCACCTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGCTCATCCCTATCTTTGATATAGAAAACTACGCACAGAAGTTCCAGGGCAGAGTCACGATTACCGCGGACGAATCCACGAGCACAGTCTATATGGAACTGAGCAGCCTGAGATCTGAGGACACGGCCATGTATTACTGTGCGAGAGGGGGGGGTTCGGGCCCTGATGTTCTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCTTCAGCTAG C<Old5 heavy chain variable region> (SEQ ID NO: 57)MDWTWRFLFVVAAATGVQSQVQLVQSGAEVKKPGSSVKVSCKASGGTFSTYAISWVRQAPGQGLEWMGGLIPIFDIENYAQKFQGRVTITADESTSTVYMELSSLRSEDTAMYYCARGGGSGPDVLDIWGQGTMVTVSSAS

The translation initiation site of the heavy chain DNA is an ATG codonwhich starts from adenine (A) at position 16 from the 5′-terminal of SEQID NO:56, and a boundary between the variable region and the constantregion of the antibody is located between adenine (A) at position 427and guanine (G) at position 428 from the 5′-terminal. In the heavy chainamino acid sequence, the heavy chain variable region is up to serine (S)residue at position 139 from the N-terminal of SEQ ID NO:57, and theconstant region is on and after alanine (A) at position 140. By a genesequence estimation software (Signal P ver. 2), the signal sequence ofheavy chain was estimated to be up to serine (S) at position 19 from theN-terminal of SEQ ID NO:57. The N-terminal of the mature form wasconsidered to be glutamine (Q) at position 20 of SEQ ID NO:57.

<Old5 light chain variable region> (SEQ ID NO: 58)CACAGATCTCTCACCATGGACATGAGGGTCCCCGCTCAGCTCCTGGGGCTCCTGCTGCTCTGGCTCCCAGGTGCCAGATGTGTCATCTGGATGACCCAGTCTCCATCCTTACTCTCTGCATCTACAGGAGACAGAGTCACCATCAGTTGTCGGATGAGTCAGGGCATTAGGAGTTATTTAGCCTGGTATCAGCAAAAACCAGGGAAAGCCCCTGAGCTCCTGATCTATGCTGCATCCACTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGTCTGAAGATTTTGCAACTTATTACTGTCAACAGTATTATAGTTTCCCGTACACTTTTGGCCAGGGGACCAAGCTGGAGATCA AACGTACGGTGG<Old5 light chain variable region> (SEQ ID NO: 59)MDMRVPAQLLGLLLLWLPGARCVIWMTQSPSLLSASTGDRVTISCRMSQGIRSYLAWYQQKPGKAPELLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQSEDFATYYCQQYYSFPYTFGQGTKLEIKRTVX

The translation initiation site of the light chain DNA is an ATG codonwhich starts from adenine (A) at position 16 from the 5′-terminal of SEQID NO:58, and a boundary between the variable region and the constantregion of the antibody is located between adenine (A) at position 402and cytosine (C) at position 403 from the 5′-terminal. In the lightchain amino acid sequence, the light chain variable region is up tolysine (K) residue at position 129 from the N-terminal of SEQ ID NO:59,and the constant region is on and after arginine (R) at position 130. Bya gene sequence estimation software (Signal P ver. 2), the signalsequence of light chain was estimated to be up to cysteine (C) atposition 22 from the N-terminal of SEQ ID NO:59. The N-terminal of themature form was considered to be valine (V) at position 23 of SEQ IDNO:59.

Each of DNA molecules encoding the heavy chain variable region and thelight chain variable region of Old17 and the amino acid sequences of theheavy chain variable region and the light chain variable region areshown in the following.

<Old17 heavy chain variable region> (SEQ ID NO: 60)GACCCGTCGACCACCATGGACTGGACCTGGAGGTTCCTCTTTGTGGTGGCAGCAGCTACAGGTGTCCAGTCCCAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGACTTCTGGAGGCACCTTCAGCAACTTTGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGTTCAACAAACTACGCACAGAAGTTCCAGGGCAGAGTCACGATTAACGCGGACGAATCCACGAGCACAGCCTACATGGAGCTGAGCAGTCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGGGTGGAGACAAATATGGTCCTTACTACTTTCACTACTGGGGCCAGGGAACCCTTGTCACCGTCTCCTCAGCTAGC<Old17 heavy chain variable region> (SEQ ID NO: 61)MDWTWRFLFVVAAATGVQSQVQLVQSGAEVKKPGSSVKVSCKTSGGTFSNFAISWVRQAPGQGLEWMGGIIPIFGSTNYAQKFQGRVTINADESTSTAYMELSSLRSEDTAVYYCAGGDKYGPYYFHYWGQGTLVTVSSAS

The translation initiation site of the heavy chain DNA is an ATG codonwhich starts from adenine (A) at position 16 from the 5′-terminal of SEQID NO:60, and a boundary between the variable region and the constantregion of the antibody is located between adenine (A) at position 432and guanine (G) at position 433 from the 5′-terminal. In the heavy chainamino acid sequence, the heavy chain variable region is up to serine (S)residue at position 139 from the N-terminal of SEQ ID NO:61, and theconstant region is on and after alanine (A) at position 140. By a genesequence estimation software (Signal P ver. 2), the signal sequence ofheavy chain was estimated to be up to serine (S) at position 19 from theN-terminal of SEQ ID NO:61. The N-terminal of the mature form wasconsidered to be glutamine (Q) at position 20 of SEQ ID NO:61.

<Old17 light chain variable region> (SEQ ID NO: 62)AGATCTCTCACCATGGACATGAGGGTCCTCGCTCAGCTCCTGGGGCTCCTGCTGCTCTGTTTCCCAGGTGCCAGATGTGACATCCAGATGACCCAGTCTCCATCCTCACTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTATTAGCAGCTGGTTAGCCTGGTATCAGCAGAAACCAGAGAAAGCCCCTAAGTCCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGCCAACAGTATAATAGTTACCCGTACACTTTTGGCCAGGGGACCAAGCTGGAGATCAAAC GTACGGT<Old17 light chain variable region> (SEQ ID NO: 63)MDMRVLAQLLGLLLLCFPGARCDIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPYTFGQGTKLEIKRTX

The translation initiation site of the light chain DNA is an ATG codonwhich starts from adenine (A) at position 19 from the 5′-terminal of SEQID NO:62, and a boundary between the variable region and the constantregion of the antibody is located between adenine (A) at position 399and cytosine (C) at position 400 from the 5′-terminal. In the lightchain amino acid sequence, the light chain variable region is up tolysine (K) residue at position 129 from the N-terminal of SEQ ID NO:63,and the constant region is on and after arginine (R) at position 130. Bya gene sequence estimation software (Signal P ver. 2), it was estimatedthat the signal sequence of light chain is up to cysteine (C) atposition 22 from the N-terminal of SEQ ID NO:63. It is considered thatthe N-terminal of the mature form is aspartic acid (D) at position 23 ofSEQ ID NO:63.

Each of DNA molecules encoding the heavy chain variable region and thelight chain variable region of Old19 and the amino acid sequences of theheavy chain variable region and light chain variable region was shown inthe following.

<Old19 heavy chain variable region> (SEQ ID NO: 64)TCGACCCCATGGACTGGACCTGGAGGTTCCTCTTTGTGGTGGCAGCAGCTACAGGTGTCCAGTCCCAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGGTGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGAGTCACGATTACCGCGGACGAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAGGACACAAATATGGCCCCTACTACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAG<Old19 heavy chain variable region> (SEQ ID NO: 65)MDWTWRFLFVVAAATGVQSQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWVGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGHKYGPYYFDYWGQGTLVTVSSASTK

The translation initiation site of the heavy chain DNA is an ATG codonwhich starts from adenine (A) at position 9 from the 5′-terminal of SEQID NO:64, and a boundary between the variable region and the constantregion of the antibody is located between adenine (A) at position 425and guanine (G) at position 426 from the 5′-terminal. In the heavy chainamino acid sequence, the heavy chain variable region is up to serine (S)residue at position 139 from the N-terminal of SEQ ID NO:65, and theconstant region is on and after alanine (A) at position 140. By a genesequence estimation software (Signal P ver. 2), it was estimated thatthe signal sequence of heavy chain is up to serine (S) at position 19from the N-terminal of SEQ ID NO:65. It is considered that theN-terminal of the mature form is the glutamine (Q) at position 20 of SEQID NO:65.

<Old19 light chain variable region> (SEQ ID NO: 66)AGATCTCTCACCATGGACATGAGGGTCCTCGCTCAGCTCCTGGGGCTCCTGCTGCTCTGTTTCCCAGGTGCCAGATGTGACATCCAGATGACCCAGTCTCCATCCTCACTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTATTAGCAGCTGGTTAGCCTGGTATCAGCAGAAACCAGAGAAAGCCCCTAAGTCCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGCCAACAGTATAATAGTTACCCTCGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAAC GTACGGTGGCT<Old19 light chain variable region> (SEQ ID NO: 67)MDMRVLAQLLGLLLLCFPGARCDIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPRTFGQGTKVEIKRTVA

The translation initiation site of the light chain DNA is an ATG codonwhich starts from adenine (A) at position 13 from the 5′-terminal of SEQID NO:66, and a boundary between the variable region and the constantregion of the antibody is located between adenine (A) at position 399and cytosine (C) at position 400 from the 5′-terminal. In the lightchain amino acid sequence, the light chain variable region is up tolysine (K) residue at position 129 from the N-terminal of SEQ ID NO:67,and the constant region is on and after arginine (R) at position 130. Bya gene sequence estimation software (Signal P ver. 2), it was estimatedthat the signal sequence of light chain is up to cysteine (C) atposition 22 from the N-terminal of SEQ ID NO:67. It is considered thatthe N-terminal of the mature form is aspartic acid (D) at position 23 ofSEQ ID NO:67.

Each of DNA molecules encoding the heavy chain variable region and thelight chain variable region of New102 and the amino acid sequences ofthe heavy chain variable region and light chain variable region wasshown in the following.

<New102 heavy chain variable region> (SEQ ID NO: 68)TCGACCACCATGGACTGGACCTGGAGGTTCCTCTTTGTGGTGGCAGCAGCTACAGGTGTCCAGTCCCAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGATCCTCGGTGAAGGTCTCCTGCATGGCTTCAGGAGGCACCGTCAGCAGCTACGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGAGATCATCCCTATCTTTGGTATAGTAAACTACGCACAGAAGTTCCAGGGCAGAGTCACGATTACCGCGGACGAATCCACGAACACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCATATATTACTGTGCGAGAGAGACAGCAGTGGCTGGTATTCTTGGTTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAG <New102 heavy chain variable region>(SEQ ID NO: 69) MDWTWRFLFVVAAATGVQSQVQLVQSGAEVKKPGSSVKVSCMASGGTVSSYAISWVRQAPGQGLEWMGEIIPIFGIVNYAQKFQGRVTITADESTNTAYMELSSLRSEDTAIYYCARETAVAGILGYWGQGTLVTVSSASTK

The translation initiation site of the heavy chain DNA is an ATG codonwhich starts from adenine (A) at position 9 from the 5′-terminal of SEQID NO:68, and a boundary between the variable region and the constantregion of the antibody is located between adenine (A) at position 423and guanine (G) at position 424 from the 5′-terminal. In the heavy chainamino acid sequence, the heavy chain variable region is up to serine (S)residue at position 138 from the N-terminal of SEQ ID NO:69, and theconstant region is on and after alanine (A) at position 139. By a genesequence estimation software (Signal P ver. 2), it was estimated thatthe signal sequence of heavy chain is up to serine (S) at position 19from the N-terminal of SEQ ID NO:69. It is considered that theN-terminal of the mature form is glutamine (Q) at position 20 of SEQ IDNO:69.

<New102 light chain variable region> (SEQ ID NO: 70)AGATCTCTCACCATGGACATGAGGGTCCTCGCTCAGCTCCTGGGGCTCCTGCTGCTCTGTTTCCCAGGTGCCAGATGTGACATCCAGATGACCCAGTCTCCATCCTCACTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTATTAGCAGCTGGTTAGCCTGGTATCAGCAGAAACCAGAGAAAGCCCCTAAGTCCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGCCAACAGTATAATAGTTACCCGTACACTTTTGGCCAGGGGACCAAGCTGGAGATCAAAC GTACGGTGGCTGCA<New102 light chain variable region> (SEQ ID NO: 71)MDMRVLAQLLGLLLLCFPGARCDIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPYTFGQGTKLEIKRTVAA

The translation initiation site of the light chain DNA is an ATG codonwhich starts from adenine (A) at position 13 from the 5′-terminal of SEQID NO:70, and a boundary between the variable region and the constantregion of the antibody is located between adenine (A) at position 399and cytosine (C) at position 400 from the 5′-terminal. In the lightchain amino acid sequence, the light chain variable region is up tolysine (K) residue at position 129 from the N-terminal of SEQ ID NO:71,and the constant region is on and after arginine (R) at position 130. Bya gene sequence estimation software (Signal P ver. 2), the signalsequence of light chain was estimated to be up to cysteine (C) atposition 22 from the N-terminal of SEQ ID NO:71. The N-terminal of themature form was considered to be aspartic acid (D) at position 23 of SEQID NO:71.

Each of DNA molecules encoding the heavy chain variable region and thelight chain variable region of Old6 and amino acid sequences of theheavy chain variable region and the light chain variable region wasshown in the following.

<Old6 heavy chain variable region> (SEQ ID NO: 72)CGACCCACCATGGAACTGGGGCTCCGCTGGGTTTTCCTTGTTGCTATTTTAGAAGGTGTCCAGTGTGAGGTGCAGTTGGTGGAGTCTGGGGGAGGCCTGGTCAAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCCATAACATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATCCATTAGTAGTAGTAGTAGTTACATATATTATGCAGACTCAGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGAGGACTGGGGCTACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGC <Old6 heavy chain variable region>(SEQ ID NO: 73) MELGLRWVFLVAILEGVQCEVQLVESGGGLVKPGGSLRLSCAASGFTFSSHNMNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREDWGYFDYWGQGTLVTVSSASTK

The translation initiation site of the heavy chain DNA is an ATG codonwhich starts from adenine (A) at position 10 from the 5′-terminal of SEQID NO:72, and a boundary between the variable region and the constantregion of the antibody is located between adenine (A) at position 417and guanine (G) at position 418 from the 5′-terminal. In the heavy chainamino acid sequence, the heavy chain variable region is up to serine (S)residue at position 136 from the N-terminal of SEQ ID NO:73, and theconstant region is on and after alanine (A) at position 137. By a genesequence estimation software (Signal P ver. 2), the signal sequence ofheavy chain was estimated to be up to cysteine (C) at position 19 fromthe N-terminal of SEQ ID NO:73. The N-terminal of the mature form wasconsidered to be glutamic acid (E) at position 20 of SEQ ID NO:73.

<Old6 light chain variable region> (SEQ ID NO: 74)AGATCTCTCACCATGGACATGAGGGTCCCCGCTCAGCTCCTGGGGCTTCTGCTGCTCTGGCTCCCAGGTGCCAGATGTGCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGGCATTAGCAGTGATTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCTCCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAATAGTTACCCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAAC GTACGGT<Old6 light chain variable region> (SEQ ID NO: 75)MDMRVPAQLLGLLLLWLPGARCAIQLTQSPSSLSASVGDRVTITCRASQGISSDLAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPFTFGPGTKVDIKRTVAA

The translation initiation site of the light chain DNA is an ATG codonwhich starts from adenine (A) at position 13 from the 5′-terminal of SEQID NO:74, and a boundary between the variable region and the constantregion of the antibody is located between adenine (A) at position 399and cytosine (C) at position 400 from the 5′-terminal. In the lightchain amino acid sequence, the light chain variable region is up tolysine (K) residue at position 129 from the N-terminal of SEQ ID NO:75,and the constant region is on and after arginine (R) at position 130. Bya gene sequence estimation software (Signal P ver. 2), the signalsequence of light chain was estimated to be up to cysteine (C) atposition 23 from the N-terminal of SEQ ID NO:75. The N-terminal of themature form was considered to be alanine (A) at position 24 of SEQ IDNO:75.

Each of DNA molecules encoding the heavy chain variable region and thelight chain variable region of 7G3 and the amino acid sequences of theheavy chain variable region and light chain variable region was shown inthe following.

<7G3 heavy chain variable region> (SEQ ID NO: 76)GTCGACCACCATGGGATGGAGCTGGATCTTTCTCTTTCTCGTGTCAGGAACTGGAGGTGTCCTCTCTGAGGTCCAGCTGCAACAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTAAAGATGTCCTGCAAGGCTTCTGGATACACCTTCACTGACTACTACATGAAGTGGGTGAAACAGAGCCATGGAAAGAGCCTTGAGTGGATTGGAGATATTATTCCTAGCAATGGTGCCACTTTCTACAACCAGAAGTTCAAGGGCAAGGCCACTTTGACTGTGGACAGATCCTCCAGCACAGCCTACATGCACCTCAACAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTACAAGATCGCATTTACTGCGGGCCTCCTGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAGCTAGC <7G3 heavy chain variable region>(SEQ ID NO: 77) MGWSWIFLFLVSGTGGVLSEVQLQQSGPELVKPGASVKMSCKASGYTFTDYYMKWVKQSHGKSLEWIGDIIPSNGATFYNQKFKGKATLTVDRSSSTAYMHLNSLTSEDSAVYYCTRSHLLRASWFAYWGQGTLVTVSAAS

The translation initiation site of the heavy chain DNA is an ATG codonwhich starts from adenine (A) at position 16 from the 5′-terminal of SEQID NO:76, and a boundary between the variable region and the constantregion of the antibody is located between adenine (A) at position 427and guanine (G) at position 428 from the 5′-terminal. In the heavy chainamino acid sequence, the heavy chain variable region is up to alanine(A) residue at position 139 from the N-terminal of SEQ ID NO:77, and theconstant region is on and after alanine (A) at position 140. By a genesequence estimation software (Signal P ver. 2), the signal sequence ofheavy chain was estimated to be up to serine (S) at position 19 from theN-terminal of SEQ ID NO:77. The N-terminal of the mature form wasconsidered to be glutamic acid (E) at position 20 of SEQ ID NO:77.

<7G3 light chain variable region> (SEQ ID NO: 78)AGATCTCACCATGGAATCACAGACTCAGGTCCTCATGTCCCTGCTGTTCTGGGTATCTGGTACCTGTGGGGACTTTGTGATGACACAGTCTCCATCCTCCCTGACTGTGACAGCAGGAGAGAAGGTCACTATGAGCTGCAAGTCTAGTCAGAGTCTGTTAAACAGTGGAAATCAAAAGAACTACTTGACCTGGTATCTGCAGAAACCAGGGCAGCCTCCTAAATTGTTGATCTATTGGGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGAACAGATTTCACTCTCACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTATTACTGTCAGAATGATTATAGTTATCCGTACACGTTCGGAGGGGGGACCAAGCTG GAAATAAAACGT<7G3 light chain variable region> (SEQ ID NO: 79)MESQTQVLMSLLFWVSGTCGDFVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYLQKPGQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYSYPYTFGGGTKLEIKR

The translation initiation site of the light chain DNA is an ATG codonwhich starts from adenine (A) at position 11 from the 5′-terminal of SEQID NO:78, and a boundary between the variable region and the constantregion of the antibody is located between adenine (A) at position 409and cytosine (C) at position 410 from the 5′-terminal. In the lightchain amino acid sequence, the light chain variable region is up tolysine (K) residue at position 133 from the N-terminal of SEQ ID NO:79,and the constant region is on and after arginine (R) at position 134. Bya gene sequence estimation software (Signal P ver. 2), the signalsequence of light chain was estimated to be up to the glycine (G) atposition 22 from the N-terminal of SEQ ID NO:79. The N-terminal of themature form was considered to be aspartic acid (D) at position 22 of SEQID NO:79.

(Preparation of Recombinant Type Antibodies)

Cells expressing a recombinant antibody were prepared by introducingeach of the constructed six recombinant antibody expression vectors intoa host cell. HEK293F (Invitrogen) was used as a host cell forexpression.

Each expression vector was introduced into HEK293F using 293Fectin(Invitrogen). The HEK293F was cultured under conditions of 5% CO₂ and37° C. using a shaker, and the culture supernatant was recovered about 5days after culturing. The recovered culture supernatant was subjected toaffinity purification using rmp Protein A (Amersham-Pharmacia Biotech).PBS as an adsorption buffer and 0.02 M of glycine buffer (pH 3) as anelution buffer, using 0.8×40 cm column (Bio-Rad Laboratories) and thelike depending on the amount for the purification. The elution fractionwas adjusted to about pH 7.2 by adding 1 M of Tris (pH 9.0). The thusprepared antibody solution was substituted to PBS using a dialysismembrane (10,000 cut, Spectrum Laboratories) and subjected tosterilization by filtration using a membrane filter MILLEX-GV having apore size of 0.22 μm (Millipore), thereby obtaining a purified humananti-IL-3Rα monoclonal antibody. The concentration of the purifiedantibody was calculated by measuring absorbance at 280 nm and regarding1 mg/ml as 1.4 OD.

A summary of amino acid sequences and SEQ ID NOs of each human antibodyCDR (complementarity-determining region) was shown in Table 1.

TABLE 1 SEQ ID NO CDR 1 CDR 2 CDR 3 CDR 1 CDR 2 CDR 3 Heavy chainvariable region Old4 113 TYAIS GIIPIFGIVNYAQKFQG GGGSGPDVLDI 114 115Old5 116 TYAIS GLIPIFDIENYAQKFQG GGGSGPDVLDI 117 118 Old17 119 NFAISGIIPIFGSTNYAQKFQG GDKYGPYYFHY 120 121 Old19 122 SYAIS GIIPIFGTANYAQKFQGGHKYGPYYFDY 123 124 New102 125 SYAIS EIIPIFGIVNYAQKFQG ETAVAGILGY 126127 Old6 128 SHNMN SISSSSSYIYYADSVKG EDWGYFD 129 130 Light chainvariable region Old4 131, 132, 133 RMSQGIRSYLA AASTLQS QQYYSFPYT Old5134, 135, 136 RMSQGIRSYLA AASTLQS QQYYSFPYT Old17 137, 138, 139RASQGISSWLA AASSLQS QQYNSYPYT Old19 140, 141, 142 RASQGISSWLA AASSLQSQQYNSYPRT New102 143, 144, 145 RASQGISSWLA AASSLQS QQYNSYPYT Old6146, 147, 148 RASQGISSDLA DASSLES QQFNSYPFT

Example 5 Purification of Anti-IL-3Rα Human Antibody from HybridomaCulture Supernatant

A hybridoma was cultured after adapting from the IL-6-containing DMEMmedium used in Example 3 to E-RDF medium (Kyokuto PharmaceuticalIndustrial Co., Ltd.). An antibody was purified from the culturesupernatant. The purification of the antibody was carried out inaccordance with Example 4.

Firstly, a hybridoma producing a human anti-IL-3Rα monoclonal antibodywas adapted to eRDF medium (Kyokuto Pharmaceutical Industrial Co., Ltd.)containing 10 ng/ml IL-6 and 10% fetal calf serum (FCS: SIGMA). Next,the obtained hybridoma was adapted to eRDF medium (KyokutoPharmaceutical Industrial Co., Ltd.) containing bovine insulin (5 μg/ml,GIBCO BRL), human transferrin (5 μg/ml, GIBCO BRL), ethanolamine (0.01mM, SIGMA), sodium selenite (2.5×10⁻⁵ mM, SIGMA) and 1% low IgG FCS(HyClone). This adapted hybridoma was cultured in a flask, and theculture supernatant was recovered. The recovered supernatant wassubjected to 10 μm and 0.2 μm filters (Gelman Sciences Inc.) to removeuseless articles such as a hybridoma and the like. The antibody waspurified from the thus recovered supernatant by the same method asExample 4.

Example 6 Calculation of Association and Dissociation Constants Usingthe Purified Anti-IL-3Rα Human Antibody

The association and dissociation constants of the purified anti-IL-3Rαantibody were analyzed using an analyzer which is based on a surfaceplasmon resonance principal (Biacore, GE Healthcare, hereinafter GE).Briefly, an anti-human antibody or anti-mouse antibody was immobilizedon a CM5 sensor tip, then an anti-IL-3Rα human or mouse antibody wasapplied thereto to allow to bind, then the soluble form of IL-3Rαprotein prepared in Example 2 was applied thereto, and the associationand dissociation were observed using Biacore 2000. Through the wholetest steps, the test method of GE Healthcare for the calculation ofassociation and dissociation constants was basically employed.

Specifically, CM5 (research grade) was used for a sensor tip (each GE).Firstly, the CM5 tip was activated by applying an equivalent mixture of400 mM EDC (N-ethyl-N′-(3-dimethylaminopropyl)carbodiimidehydrochloride)and 100 mM NHS (N-hydroxysuccinimide) to the CM5 tip. Next, an antibodyto the human antibody attached to the Human Antibody Capture Kit (GE)(hereinafter, referred to as anti-human antibody antibody) was dilutedwith the solution attached to the kit and applied thereto, therebyimmobilizing the required amount of the anti-human antibody antibody tothe CM5 tip. Regarding the mouse antibody to be used as a control, anantibody to the mouse antibody attached to the Mouse Antibody CaptureKit (GE) (hereinafter, referred to as anti-mouse antibody antibody) wasdiluted with the solution attached to the kit and applied thereto,thereby immobilizing a necessary amount thereof to the CM5 tip. Next,the surface of the activated tip was blocked and inactivated by applying1 M of ethanolamine dihydrochloride thereto. By the above steps untilthis, preparation of a CM5 sensor tip which can measure the dissociationconstant K_(D) was completed.

Next, each of anti-IL-3Rα antibodies, was diluted to give aconcentration of 5 μg/ml with HBS-FP buffer (GE) one kind per one flowcell and applied, thereby allowing it to bind to the immobilizedanti-human antibody antibody or anti-mouse antibody antibody. Next, thesoluble form of IL-3Rα protein was applied thereto. In order todissociate the bound anti-IL-3Rα antibody and soluble form of IL-3Rαprotein, 3 M MgCl₂ attached to Human Antibody Capture Kit or pH 1.7 ofGlycine-HCl attached to Mouse Antibody Capture Kit was applied in theamount attached to the kit. The steps until this were regarded as onestep. By repeating the same steps using two or more concentrations ofthe soluble form of IL-3Rα protein, data for calculating association anddissociation constants (sensorgram) were obtained.

The concentration of the soluble form of human IL-3Rα protein applied toa subject was calculated as described in Example 2 by measuring theabsorbance at 280 nm and regarding 1 mg/ml as 1.4 OD. The molecularweight of the soluble form of human IL-3Rα protein was calculated asfollows. Regarding molecular weight of human IL-3Rα protein, it has beenreported that it comprises 360 amino acid residues, has six N-type sugarchain binding sites and the molecular weight is 70 kDa (The CytokineFacts Book second edition, Academic Press). Accordingly, the molecularweight of the soluble form of human IL-3Rα protein was calculated asabout 63 kDa by subtracting molecular weights of the amino acids of thetransmembrane region and the intracellular region from 70 kDa known as areference information and adding, to the resulting value, the molecularweight of the amino acids of the Flag sequence.

In the analysis, Biaevaluation software (GE) was used and BiaevaluationSoftware Handbook was referred. Specifically, by carrying outsimultaneous analysis of kinetics analysis, employing basically the 1:1Langmuir binding reaction model and fitting, association rate constant(Ka) and dissociation rate constant (Kd) were calculated, and the valueof dissociation constant K_(D) was calculated by the calculation ofKd/Ka.

The results are shown in the following table 2.

TABLE 2 Antibody name Ka Kd K_(D) Human antibodies Old4 3.88 × 10⁵ 5.15× 10⁻⁴ 1.33 × 10⁻⁹ Old5 7.17 × 10⁵ 4.72 × 10⁻⁴  6.58 × 10⁻¹⁰ Old17 2.08× 10⁵ 2.98 × 10⁻⁴ 1.43 × 10⁻⁹ Old19 1.54 × 10⁵ 4.99 × 10⁻⁴ 3.24 × 10⁻⁹New102 6.02 × 10⁵ 4.80 × 10⁻⁴  7.98 × 10⁻¹⁰ Old6 1.71 × 10⁶ 2.15 × 10⁻⁵1.26 × 10⁻⁹ Chimeric antibody 7G3 2.48 × 10⁵ 4.66 × 10⁻⁴ 1.88 × 10⁻⁹Mouse antibodies 7G3 1.68 × 10⁵ 9.52 × 10⁻⁵  5.66 × 10⁻¹⁰ 9F5 7.13 × 10⁴ 6.5 × 10⁻⁵  9.11 × 10⁻¹⁰ 107D2.08 4.16 × 10⁵ 2.03 × 10⁻⁵ 4.88 × 10⁻⁸AC145 7.66 × 10⁴ 4.26 × 10⁻⁵ 5.57 × 10⁻⁸ L-16 8.13 × 10⁵ 4.16 × 10⁻⁵5.12 × 10⁻⁹

Example 7 Epitope Analysis of Anti-Human IL-3Rα Human Antibody(Preparation of IL-3Rα/GM-CSFRα Chimeric Protein Expression Cell)

In order to carry out epitope analysis of IL-3Rα antibody, a chimericprotein in which a portion of the extra-membrane region of IL-3Rα wasreplaced by GM-CSFRα was expressed in a cell, and binding activity ofeach anti-IL-3Rα antibody to the cell was analyzed. In brief, firstly,the IL-3Rα molecule and GM-CSFRα molecule were divided into threeregions (A, B and C domains from the above-mentioned N-terminal),secondly vectors which express molecules in which each of the A, B and Cdomains of the IL-3Rα molecule was replaced by the corresponding domainof GM-CSFRα molecule were respectively constructed, thirdly, these wereforcedly expressed in HEK293F cell, and fourthly, whether or not eachanti-IL-3Rα antibody labeled with a fluorescence dye binds thereto wasobserved by flow cytometry.

(Preparation of GM-CSFR/pEF6/Myc-HisC Plasmid DNA)

A cDNA of human GM-CSFR receptor a chain (GM-CSFRα, CD116) was amplifiedfrom a spleen-derived cDNA (CLONTECH Human MTC Panel) by a PCR methodusing KOD-Plus-Ver. 2 (Toyobo Co., Ltd.). As a PCR device, GeneAmp PCRSystem 9700 (Applied Biosystems) was used. Regarding the PCR, after adenaturation step at 94° C. for 2 minutes, a three step reaction at 98°C. 10 seconds-55° C. 30 seconds-68° C. 75 seconds was carried out 35cycles. The PCR primers used are as follows.

hCD116Fw-MfeI: (SEQ ID NO: 80)5′-CGGCAATTGCCACCATGCTTCTCCTGGTGACAAGCCT-3′ hCD116Rv-NotI:(SEQ ID NO: 81) 5′-ATTGCGGCCGCTCAGGTAATTTCCTTCACGG-3′

The thus obtained PCR products were subjected to 0.8% agarose gelelectrophoresis (TAE buffer). DNA was visualized by ethidium bromidestaining. A band of at around 1.2 kb was cut out, and the DNA wasextracted using JETsorb Kit (Genomed, Bad Oeynhausen, Germany) and thendigested with NotI and MfeI. A pEF6/Myc-His C plasmid DNA (Invitrogen)was digested with EcoRI and NotI. Each DNA was subjected to 0.8% agarosegel electrophoresis and bands of at around 1.2 kb and around 6 kb werecut out, and the DNA molecules were extracted using JETsorb Kit(Genomed, Bad Oeynhausen, Germany). Then, 0.5 μl of a pEF6/Myc-His Cplasmid DNA-derived DNA solution and 4 μl of a PCR product-derived DNAsolution were mixed and subjected to ligation using TaKaRa Ligation Kit(TAKARA BIO INC.). Regarding the transformation, a ligation sample andDH5 alpha competent cells were mixed and spread on an LB plate.Insertion check was carried out by colony direct PCR using LA Taq(TAKARA BIO INC.). Regarding the PCR, after a denaturation step at 94°C. for 5 minutes, a three step reaction at 94° C. 30 seconds-55° C. 30seconds-72° C. 2 minutes was carried out 40 cycles and then a treatmentat 99° C. for 30 minutes was carried out.

The PCR primers used were as follows.

hCD116Fw-MfeI: (SEQ ID NO: 82)5′-CGGCAATTGCCACCATGCTTCTCCTGGTGACAAGCCT-3′ hCD116Rv-NotI:(SEQ ID NO: 83) 5′-ATTGCGGCCGCTCAGGTAATTTCCTTCACGG-3′

The thus obtained PCR products were subjected to 0.8% agarose gelelectrophoresis (135 V, 15 minutes, TAE buffer). DNA was visualized byethidium bromide staining. Using a colony from which amplification ofaround 1.2 kb was obtained, nucleotide sequence was determined by adirect sequencing method. In the reaction of sequence samples, BigDye®Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) and GeneAmpPCR System 9700 (Applied Biosystems) were used (these were used in theall DNA sequence analyses in this specification). The PCR primers usedare as follows.

hCD116Rv-NotI: (SEQ ID NO: 85) 5′-ATTGCGGCCGCTCAGGTAATTTCCTTCACGG-3′hCD116SeqFw1: (SEQ ID NO: 86) 5′-TGAACTGTACCTGGGCGAGG-3′ hCD116SeqFw2:(SEQ ID NO: 87) 5′-CTGGCACGGAAAACCTACTG-3′ hCD116SeqRv1: (SEQ ID NO: 88)5′-CCTGAATTTGGATAAAGCAG-3′

ABI 3700XL DNA analyzer (Applied Biosystems) was used as a sequenceanalyzing device (this was used in the all DNA sequence analyses in thisspecification). By selecting a clone in which mutation in the amino acidsequence by PCR was not found, plasmid DNA was extracted by Largeprepmethod (QIAGEN).

(Preparation of IL-3RA-FLAG/pEGFP-N1)

The full length cDNA of human IL-3Rα (CD123) was amplified by PCR andFLAG tag was linked to its downstream (IL-3RA-FLAG/pEGFP-N1).

Human IL-3RA cDNA was amplified by a PCR method using hCD123/pEGFP-N1plasmid DNA as a template and LA Taq (TAKARA BIO INC.). Regarding thePCR, after a denaturation step at 95° C. for 30 seconds, a three stepreaction at 95° C. 15 seconds-56° C. 15 seconds-72° C. 60 seconds wascarried out 10 cycles and then 2 minutes of an elongation reaction wascarried out. The PCR primers used are as follows.

T7: (SEQ ID NO: 89) 5′-TAATACGACTCACTATAGGG-3′ hCD123-C-FLAG-R1:(SEQ ID NO: 90) 5′-TCGTCATCGTCCTTGTAGTCAGTTTTCTGCACGACCTGTA-3′

Using 2 μl of the PCR product as a template, amplification was carriedout by a PCR method using LA Taq (TAKARA BIO INC.). Regarding the PCR,after a denaturation step at 95° C. for 1 minute, a three step reactionat 95° C. 15 seconds-56° C. 15 seconds-72° C. 60 seconds was carried out15 cycles and then an elongation reaction at 72° C. for 2 minutes wascarried out. The PCR primers used are as follows.

IL-3Rα_Fw: (SEQ ID NO: 91) 5′-CGGCAATTGCCACCATGGTCCTCCTTTGGCTCAC-3′C-FLAG-NotR2: (SEQ ID NO: 92)5′-AAAAGCGGCCGCTCACTTGTCGTCATCGTCCTTGTAGTC-3′

The thus obtained PCR products were subjected to 0.8% agarose gelelectrophoresis (135 V, 15 minutes, TAE buffer). DNA was visualized byethidium bromide staining. A band of at around 1 kb was cut out, and theDNA was extracted using Wizard SV Gel and PCR Clean-Up System. The wholeamount of the extracted DNA was digested with MfeI and NotI andsubjected to 0.8% agarose gel electrophoresis (135 V, 15 minutes, TAEbuffer). DNA was visualized by ethidium bromide staining. A band of ataround 1 kb was cut out, and the DNA was extracted using Wizard SV Geland PCR Clean-Up System. Then, 5 μl of the thus extracted IL-3RA-FLAGcDNA and 1 μl of the pEGFP-N1 plasmid DNA which had been cleaved withEcoRI and NotI were mixed and ligated using TaKaRa Ligation Kit (TAKARABIO INC.). Regarding the transformation, a ligation sample and DH10Bcompetent cells were mixed and spread on an LB plate (containingkanamycin). Insertion check was carried out by colony direct PCR usingLA Taq (TAKARA BIO INC.). Regarding the PCR, after a denaturation stepat 95° C. for 1 minute, a three step reaction at 95° C. 15 seconds-56°C. 15 seconds-72° C. 60 seconds was carried out 35 cycles and then anelongation reaction at 72° C. for 2 minutes was carried out. The PCRprimers used are as follows.

pEGFP-N1-Fw: (SEQ ID NO: 93) 5′-CGTGTACGGTGGGAGGTCTA-3′ pEGFP-N1-Re:(SEQ ID NO: 94) 5′-TTTATGTTTCAGGTTCAGG-3′

A plasmid DNA was extracted by a Miniprep method from a colony in whichamplification of around 0.8 kb was obtained.

It was found by a DNA sequence analysis that the purifiedIL-3RA-FLAG/pEGFP-N1 plasmid DNA does not have a mutation caused by thePCR and that the FLAG tag is present therein. The primers used in theDNA sequence analysis are as follows.

pEGFP-N1-Fw: (SEQ ID NO: 95) 5′-CGTGTACGGTGGGAGGTCTA-3′ pEGFP-N1-Re:(SEQ ID NO: 96) 5′-TTTATGTTTCAGGTTCAGG-3′

(Domain Mapping of IL-3Rα)

As a result of BLASTP search (database: Protein Data Bank proteins(pdb)), IL-13 receptor alpha chain (IL-13Rα) was hit with the highestscore (PDB: 3BPNC; Chain C, Crystal Structure of the Il4-Il4r-Il13raTernary Complex). Using the PDB file down-loaded from Protein Data Bankand a graphic software RasMol, three-dimensional structure of theIL-13Rα protein was visualized and three domains constituting theextracellular region (the above-mentioned A, B and C domains) weredivided. Using a Multiple Alignment software MUSCLE, IL-3Rα amino acidsequence and IL-13Rα amino acid sequence were compared and IL-3Rαextracellular region was also divided into three domains. Further,GM-CSFRα and IL-3Rα were compared in the same manner and GM-CSFRαextracellular region was also divided into three domains.

In order to assign epitopes of anti-human IL-3Rα human antibodies,proteins in which each of the three domains of IL-3Rα was replaced oneby one by said domains of GM-CSFRα were prepared and expressed on thecell membrane and the presence or absence of antibody binding wasconfirmed.

Using the IL-3RA-FLAG/pEGFP-N1 plasmid DNA as a template, amplificationwas carried out by a PCR method which uses PrimeSTAR® HS DNA Polymerase(TAKARA BIO INC.). Regarding the PCR, a two step reaction at 98° C. 10seconds-68° C. 6 minutes was carried out 25 cycles. The PCR primers usedare as follows.

A domain deficiency; CD123R11pEGFPN1: (SEQ ID NO: 97)AAAGGTACCGAATTCGAAGCTTGAGCTC CD123F11: (SEQ ID NO: 98)AAAGGTACCGGGAAGCCTTGGGCAGGT B domain deficiency; CD123R12-2:(SEQ ID NO: 99) AAAGGTACCACTGTTCTCAGGGAAGAGGAT CD123F12-2:(SEQ ID NO: 100) AAAGGTACCCAGATTGAGATATTAACTCC C domain deficiency;CD123R13: (SEQ ID NO: 101) AAAGGTACCTGAAAAGACGACAAACTT CD123F13:(SEQ ID NO: 102) AAAGGTACCTCGCTGCTGATCGCGCTG

The thus obtained PCR product was subjected to 0.8% agarose gelelectrophoresis (135 V, 15 minutes, TAE buffer). DNA was visualized byethidium bromide staining. After confirming the amplification, this waspurified using Wizard SV Gel and PCR Clean-Up System. The thus obtainedDNA was digested with KpnI and DpnI, purified using Wizard SV Gel andPCR Clean-Up System and ligated using TaKaRa Ligation Kit. Regarding thetransformation, a ligation sample and DH10B competent cells were mixedand spread on an LB plate (containing kanamycin). Insert check wascarried out by colony direct PCR using LA Taq (TAKARA BIO INC.).Regarding the PCR, after a denaturation step at 95° C. for 1 minute, athree step reaction at 95° C. 15 seconds-56° C. 15 seconds-72° C. 40seconds was carried out 38 cycles and then an elongation reaction at 72°C. for 2 minutes was carried out. The PCR primers used are as follows.

pEGFP-N1-Fw: (SEQ ID NO: 103) 5′-CGTGTACGGTGGGAGGTCTA-3′ pEGFP-N1-Re:(SEQ ID NO: 104) 5′-TTTATGTTTCAGGTTCAGG-3′

The thus obtained PCR product was subjected to 0.8% agarose gelelectrophoresis (135 V, 15 minutes, TAE buffer). DNA was visualized byethidium bromide staining. A plasmid DNA was extracted by the Miniprepmethod from a colony in which amplification at around 1 kb was obtained.

Using the GM-CSFR/pEF6/Myc-His C plasmid DNA as a template,amplification was carried out by a PCR method which uses PrimeSTAR® HSDNA Polymerase (TAKARA BIO INC.). Regarding the PCR, a two step reactionat 98° C. 10 seconds-68° C. 30 seconds was carried out 25 cycles. ThePCR primers used are as follows.

A domain insertion; GM-CSFRF11: (SEQ ID NO: 105)AAAGGTACCGCCACCATGCTTCTCCTGGTGACA GM-CSFRR11: (SEQ ID NO: 106)AAAGGTACCTGAATTTGGATAAAGCAG B domain insertion; GM-CSFRF12:(SEQ ID NO: 107) AAAGGTACCGGAAGGGAGGGTACCGCT GM-CSFRR12:(SEQ ID NO: 108) AAAGGTACCCTTTGTGTCCAAAAGTGA C domain insertion;GM-CSFRF13: (SEQ ID NO: 109) AAAGGTACCAAAATAGAACGATTCAAC GM-CSFRR13:(SEQ ID NO: 110) AAAGGTACCAATGTACACAGAGCCGAG

The thus obtained PCR product was subjected to 0.8% agarose gelelectrophoresis (135 V, 15 minutes, TAE buffer). DNA was visualized byethidium bromide staining. After confirming the amplification, this waspurified using Wizard SV Gel and PCR Clean-Up System.

The thus obtained DNA was digested with KpnI and then purified usingQIAquick Gel Extraction Kit (QIAGEN), mixed with IL-3RA-FLAG/pEGFP-N1plasmid DNA in which the corresponding domain was deleted (alreadycleaved with KpnI and purified) and ligated using TaKaRa Ligation Kit.Regarding the transformation, a ligation sample and DH10B competentcells were mixed and spread on an LB plate (containing kanamycin).Insert check was carried out by colony direct PCR using LA Taq (TAKARABIO INC.). Regarding the PCR, after a denaturation step at 95° C. for 1minute, a three step reaction at 95° C. 15 seconds-56° C. 15 seconds-72°C. 40 seconds was carried out 38 cycles and then an elongation reactionat 72° C. for 2 minutes was carried out. The PCR primers used are asfollows.

pEGFP-N1-Fw: (SEQ ID NO: 111) 5′-CGTGTACGGTGGGAGGTCTA-3′ pEGFP-N1-Re:(SEQ ID NO: 112) 5′-TTTATGTTTCAGGTTCAGG-3′

The thus obtained PCR product was subjected to 0.8% agarose gelelectrophoresis (135 V, 15 minutes, TAE buffer). DNA was visualized byethidium bromide staining. A plasmid DNA was extracted by the Miniprepmethod from a colony in which amplification at around 1 kb was obtained.

(Labeling of Anti-IL-3Rα Human Antibody with Fluorescence Dye)

In order to determine binding activity of anti-human IL-3Rα humanantibodies, each human antibody was labeled with a fluorescence dyeAlexaFlour488 (Molecular Probe, Invitrogen). Regarding the labelingmethod, it was carried out in accordance with the manual provided byInvitrogen, and regarding the detection, fluorescence was detected byFL1 of a flow cytometry (FACS Calibur, BD Biosciences).

Specifically, 1/10 volume of 1 M Na₂CO₃ was added to an antibodysolution dissolved in PBS. Next, a predetermined amount of the antibodysolution described in the manual was added to a container containingpowder of AlexaFlour488 to which tetrafluorophenyl (TFP) had been added,and allowed to undergo the reaction in the dark at room temperature for1 hour while stirring. Next, after a gel filtration column (NAP-10 andthe like, GE Healthcare) was sufficiently substituted with PBS, thesolution of antibody reacted with AlexaFlour488 was added thereto whilethe buffer of the antibody solution was substituted with PBS. Anantibody fraction which showed yellow green was obtained. Regarding theAlexaFlour488-labeled anti-human IL-3Rα antibody obtained in the abovemanner, the absorbances at wavelengths of 280 nm and 494 nm (A280 andA494, respectively) were measured using a spectrometer, and the antibodyconcentration was calculated by the following calculation formula.

Antibody concentration (mg/ml)=(A280−A494×0.11)/1.4

(Flow Cytometry Analysis of IL-3Rα/GM-CSFRα Chimeric Protein ExpressionCell Using Labeled Anti-IL-3Rα Antibody)

HEK293T cell (ATCC CRL 1268) was used for the preparation of theIL-3Rα/GM-CSFRα chimeric protein expression cell. Using 293Fectin(Invitrogen), the plasmid DNA obtained in the above was introduced as anexpression vector into the HEK293T. The HEK293T to which the expressionvector was introduced was cultured using a shaker under conditions of 5%CO₂ and 37° C. after 2 days of the introduction, the obtained proteinwas used for in the flow cytometry analysis.

From 100,000 to 1,000,000 cells of the chimeric protein expression cellwere allowed to react for 30 minutes on ice with at a concentration of 1μg/ml with an AlexaFlour488-labeled human antibody or a commerciallyavailable FITC-labeled anti-IL-3Rα mouse antibody (7G3 or 9F5: both fromBD Biosciences, 6H6: Acris Antibodies, AC145: Milteny Biotech, 107D2.08:Dendritics). A staining medium (Dulbecco's PBS supplemented with 2%fetal bovine serum, 2 mM EDTA and 0.05% NaN₃) was used for the dilutionof antibodies and cells. Next, the cells reacted with antibodies werewashed three times with the staining medium and whether the labeledantibody bound to the cell was confirmed by flow cytometry.

The results are shown in FIGS. 1 and 2. Reactions of 7G3, 9F5, 6H6 andAC145 antibodies disappeared only in the cells expressing a protein inwhich A domain was replaced by GM-CSFRα. On the other hand, reactions ofOld4, Old5, Old19 and New102 antibodies disappeared in the cellsexpressing a protein in which B domain was replaced by GM-CSFRα.Regarding Old19, its reaction with the cells expressing a protein inwhich A domain was replaced by GM-CSFRα also disappeared. Regarding theOld6 and 107D2.08, their reaction to B domain- and C domain-substitutedprotein expression cells disappeared.

Based on the above, it was shown a possibility that 7G3, 9F5, 6H6 andAC145 recognized A domain, and Old4, Old5 and New102 recognized Bdomain, Old19 recognized A domain and B domain, and Old6 and 107D2.08recognized B domain and C domain. Accordingly, the reactivity of variousanti-IL-3Rα antibodies to A to C domains of IL-3Rα was as the followingTable 3.

TABLE 3 7G3 9F5 6H6 AC145 107D2.08 Old19 New102 Old4 Old5 Old6 Old28 No++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ substitution A domain − − − − ++ − ++++ ++ ++ + substitution B domain ++ ++ ++ + − − − − − − − substitution Cdomain ++ ++ ++ ++ − ++ ++ ++ ++ − ++ substitution

Example 8 Analysis of IL-3 Signaling Blocking Activity of Anti-IL-3RαAntibodies

In order to examine whether the thus obtained IL-3Rα antibodies inhibitIL-3 signaling, a cell line TF-1 (DMSZ no. ACC344) which grows IL-3- orGM-CSF-dependently was used. Specifically, the TF-1 cell was dilutedwith RPMI 1640 medium containing 1 ng/ml of IL-3 and 10% fetal calfserum (TF-1 medium) and dispensed on a 96-well plate. Further, variousIL-3Rα antibodies and a human serum-derived IgG as a negative controlantibody were diluted with the TF-1 medium, transferred to the 96-wellplate and added in such a manner that final concentration of eachantibody gave a final concentration of 10 or 100 μg/ml. As a control, awell of cell-free medium alone and a well to which the TF-1 cell wasadded were prepared. After 3 days of culturing under an environment of37° C. and 5% CO₂, CelltiterGlo (Promega) was added thereto in an amountequivalent to the medium. After 30 minutes of still standing, theemission was determined using a plate reader (ARBO, Perkin Elmer).

For the growth inhibition ratio, the following calculation was carriedout.

(emission of sample−well with no cells)/(well to which TF-1 cell alonewas added−well with no cells)×100(%)

Regarding the commercially available antibodies 9F5, 6H6 and 107D2.08,an NAP-5 column was used for the purpose of substituting the buffer withPBS. Specifically, 0.5 ml of an antibody solution was added to the NAP-5column sufficiently substituted with PBS. Next, by adding 1.0 ml of PBS,the solution discharged from the column was recovered. By carrying outsterilization by filtration using a membrane filter MILLEX-GV(Millipore) having a pore size of 0.22 μm, an antibody dissolved in PBSas a solvent was obtained.

The results are shown in FIG. 5. It was found that the antibody Old4,antibody Old5, antibody Old17, antibody Old19, antibody New102, antibody9F5 and antibody 6H6 did not inhibit the IL-3 signaling, and on theother hand, it was found that the antibody 7G3, antibody Old6 andantibody 107D2.08 inhibited IL-3 signaling.

Example 9 Examination of Influence Upon Colony Forming Ability UsingAnti-IL-3Rα Human Antibody

A colony assay was carried out to find whether various IL-3Rα antibodieshave effects upon the colony forming ability by hematopoietic precursorcells.

In brief, 400 cells/ml of cord blood-derived CD34 positive cell(AllCells) was added to a Methocult medium (Stem Cell Technologie)supplemented with erythropoietin, IL-3, G-CSF and Stem Cell Factor, andthe number of colonies was measured 14 days to 16 days thereafter. Thecolonies were counted by dividing them into a granulocyte/macrophagesystem colony (CFU-GM), an erythroid system colony (BFU-E) and mixedcolonies (CFU-Mix or CFU-GEMM). Regarding the classification method ofcolony types, a manual provided by Stem Cell Technologie or varioustextbooks on hematology was used as references.

As the antibodies, each of the chimeric 7G3 antibody as an antibody inwhich IL-3 signaling blocking activity was found in Example 8 and theNew102 antibody as an antibody in which the blocking activity was notfound was used.

The results are shown in FIG. 6. In the colony assay in whicherythropoietin, IL-3, G-CSF and Stem Cell Factor were added, decrease inthe number of colonies and decrease in the colony size were found by theaddition of the 7G3 antibody which had the blocking activity of IL-3signaling. On the other hand, change in the number of colonies by theaddition of the New102 antibody was not found. Based on this result, itis considered that the influence upon the normal hematopoietic functionis small and side effects are few when the IL-3 signaling is notinhibited or blocked.

Example 10 Antitumor Effect in a Mouse Tumor Bearing Model UsingAnti-IL-3Rα Human Antibody

The thus obtained anti-IL-3Rα antibody was administered to mouse tumorbearing model and its antitumor effect was examined. In brief, aleukemia cell was transferred into a mouse through its caudal vein, theantibody was administered thereto on the next day, and about 3 weeksthereafter, the number of leukemia cells in bone marrow cells collectedfrom a bone of the mouse was counted.

Specifically, 0.01 ml equivalent of anti-asialoGM1 antiserum (Wako PureChemical Industries, Ltd.) was diluted with physiological saline andadministered to SCID mouse (CLEA Japan Inc.) (Day −1). On the next day,500,000 cells of a cell line of acute myeloid leukemia, MOLM13 (ATCC),were transplanted through caudal vein (Day 0). Further on the next day(Day 1), 1014 of the anti-IL-3Rα antibody was intraperitoneallyadministered. The mouse was sacrificed on Day 21, bone marrow wascollected from thighbones and shinbones and the bone marrow cells werestained with FITC-labeled human CD45 antibody and PE-labeled anti-IL-3Rα(both from BD Biosciences). Specifically, the antibody was added toabout 1,000,000 cells of the bone marrow cell, to give a finalconcentration of 1 μg/ml for each and allowed to stand still on ice for30 minutes under shade. Thereafter, the cells stained with the antibodywas washed 3 times using a staining medium (a solution prepared byadding 2% fetal bovine serum, 0.05% sodium azide and 2 mM EDTA to PBS(GIBCO)), and a human CD45 positive and human IL-3Rα positive cell wasdetected by a flow cytometry (FACSCalibur, BD Biosciences). Also, at thetime of collecting mouse bone marrow, the number of bone marrow cellswas counted using TURK solution. Further, the absolute number of theMOLM13 cells contained in one thighbone was counted by simultaneouslyadding quantified fluorescent beads (Flow-Count, Beckman Coulter) at thetime of the above-mentioned antibody staining.

The results are shown in FIG. 7. It was found that the number of MOLM13cells in the thighbone bone marrow in each antibody administered groupis markedly reduced in comparison with the Vehicle group to which theantibody was not administered. This result shows that the anti-IL-3Rαantibody has a possibility as a therapeutic agent for leukemia.

Example 11 Toxicity Test of IL-3Rα Expression Cell by Anti-IL-3RαAntibody

In order to measure antibody-mediated cytotoxicity (antibody-dependentcellular cytotoxicity, hereinafter referred to as ADCC), this wascarried out in the presence of antibody using a human peripheral bloodmononuclear cells (peripheral blood mononuclear cells, hereinafter PBMC)as an effector.

Peripheral blood was collected from a healthy volunteer and ananticoagulant was added thereto. The blood was put statically onFicoll-Plaque Plus (GE Healthcare) such that the interface was notdisturbed and centrifuged at 2,000 rpm for 20 minutes using a largecentrifuge (CF9RX, Hitachi, Ltd.) The intermediate layer containing thecells was collected and washed with PBS, platelets were removed bycentrifugation at 900 rpm for 20 minutes, and the peripheralblood-derived mononuclear cells (PBMC) were used as an effector.

Further, PBMC cultured overnight under conditions of 37° C. and 5% CO₂using RPMI 1640 medium containing 10% fetal bovine serum to which humanIL-2 (Peprotech) was added to a final concentration of 4 ng/ml (40 IU/mlor more) was also used as an effector of ADCC assay.

In the method, in simple, a target cell is cultured in the presence ofan antibody and PBMC and the lysis rate of specific target cell by theantibody is measured.

The following “Colon-26/hCD123 ADCC assay method” was used for themeasurement of the lysis rate. Specifically, a target cell was labeledwith ⁵¹Cr by culturing the IL-3Rα forced expression Colon-26 cell as atarget cell at 37° C. for 1 hour in the presence of 5% CO₂, togetherwith sodium chromate labeled with a radioisotope ⁵¹Cr (Na₂ ⁵¹CrO₄,Perkin Elmer, NEZ030S). The labeled target cell was washed 3 times toremove excess ⁵¹Cr and then suspended in the medium and transferred to a96-well plate to which antibodies had been added in advance at variousconcentrations. PBMC was suspended in the medium and transferred to theplate to which the target cell and antibodies had been added(effector/target ratio=100). As the antibodies, the anti-IL-3Rα antibodypurified in Example 4 was used, and human serum-derived IgG (SIGMA) as anegative control. As various controls, a well of the medium and targetcell alone, a well of PBMC and target alone and a well supplemented withTriton-X were prepared. The 96-well plate filled with mixed solutionswas cultured at 37° C. for 4 hours in the presence of 5% CO₂.

After centrifugation of the plate, 50 μl of each supernatant wastransferred to a scintillator-containing 96-well plate (Lumaplate™,Perkin Elmer) and dried at 56° C. for 2 hours. The plate was sealed(TopSeal-A, Packard) and measured using a microplate reader (TopCount,Perkin Elmer).

Regarding the lysis rate of the target cell, amount of ⁵¹Cr in thesodium chromate released into the medium due to the lysis of cells wasmeasured. That is, the “specific lysis rate” was calculated by dividinga value obtained by subtracting the value of a well to which theantibody was not added from the value of each well, by a value obtainedby subtracting the value of a well to which the antibody was not addedfrom the value of a well to which Triton-X was added (specific lysisrate is set to 100%).

The results are shown in FIG. 8 and FIG. 9. In each IL-3Rα antibodies,the ADCC activity for the target cell was found depending on theconcentration. Also, these antibodies exhibited higher ADCC activitythan the chimeric 7G3 antibody as a control. This shows that the IL-3Rαantibody exhibit high ADCC activity for IL-3Rα expression cells and hasa possibility of a treatment in which drug efficacy is a removal of theIL-3Rα positive cell.

Example 12 Affinity Test of Anti-IL-3Rα Antibody to Monkey IL-3RαProtein

Regarding the presence or absence of binding of the thus obtainedanti-human IL-3Rα antibody to monkey IL-3Rα, whether or not theanti-human IL-3Rα antibody prepared in Example 7 binds to the Macacafascicularis IL-3Rα forced expression cell prepared in Example 1 wasanalyzed using flow cytometry.

Specifically, 2×10⁵ cells of the monkey IL-3Rα forced expression L929cell were allowed to react with 100 μl so that a final concentration of10 μg/ml, of the anti-human IL-3Rα antibody at 4° C. for 30 minutes. Theantibodies used are anti-dinitrophenol (DNP) human IgG1 antibody(manufactured by this firm) as a negative control and Old4, Old5, Old17,Old19, New102 and chimeric 7G3 antibodies. Thereafter, this was washed 3times using a staining medium (Dulbecco's PBS supplemented with 2% fetalbovine serum, 2 mM EDTA and 0.05% NaN₃). Next, a PE-labeled anti-humanantibody λ chain specific antibody (Southern Bio) was allowed to undergothe reaction in the staining medium at a final concentration of 1 μg/mland washed 3 times with the staining medium in the same manner. Finally,the cells were mixed with the staining medium and whether the presenceor absence of PE positive was analyzed by flow cytometry.

The results are shown in FIG. 10. It was found that the anti-humanIL-3Rα human antibodies, Old4, Old5, Old17, Old19, New102 and chimeric7G3 antibodies, react with the Macaca fascicularis IL-3Rα.

Example 13 Detailed Epitope Analysis of Anti-Human IL-3Rα HumanAntibodies (Preparation of IL-3Rα/GM-CSFRα Chimeric Protein ExpressionCell)

In order to carry out further detailed epitope analysis of IL-3Rαantibodies, a chimeric protein in which a region smaller than the domainof IL-3Rα extra-membrane region was replaced by GM-CSFRα was expressedin a cell and affinity of each anti-IL-3Rα antibody to the cell wasanalyzed. In brief, firstly, a region considered to be positioned atoutside based on a three dimensional structure prediction of IL-3Rαmolecule was determined, secondly, vectors which express IL-3Rαmolecules in which the small region was replaced by GM-CSFRα wererespectively constructed, thirdly, these were forcedly expressed inHEK293F cell and fourthly, whether or not each anti-IL-3Rα antibodylabeled with fluorescence dye binds thereto was observed by flowcytometry.

(Domain Mapping of IL-3Rα)

Among the 3 domains divided according to Example 7, A and B domainswhich were recognized by the obtained antibodies Old19 and New102 wereselected and analyzed in detail. Based on the three dimensionalstructure of IL-4 receptor alpha chain (IL-4Rα, CD124) (PDB: 3BPNC;Chain C, Crystal Structure of the II4-II4r-II3ra Ternary Complex), threedimensional structure of IL-3Rα was subjected to homology modeling usingSWISS-MODEL (http://swissmodel.expasy.org//SWISS-MODEL.html). Thepredicted IL-3Rα protein structure was visualized using a graphicsoftware RasMol (http://rasmol.org/) and 7 regions considered to bepositioned at extracellular amino acid region of IL-3Rα molecule weredetermined (FIG. 4).

In order to specify epitope of anti-human IL-3Rα human antibody, aprotein in which corresponding regions of GM-CSFRα were replaced by the6 regions of IL-3Rα divided as described in the above was prepared andexpressed on the cell membrane, and the presence or absence of bindingof antibodies was determined.

Using the IL-3RA-Flag/pEGFP-N1 plasmid DNA as a template, amplificationwas carried out by a PCR method which uses PrimeSTAR® HS DNA polymerase(TAKARA BIO INC.). Regarding the PCR, a two step reaction at 98° C. 10seconds-68° C. 5 minutes was carried out 25 cycles. The PCR primers usedare as follows.

Region 1 Deficiency; CD123-Fw21: (SEQ ID NO: 149)CGTGGAACCCGCAGTGAACAATAGCTATT CD123-Re21: (SEQ ID NO: 150)ACTCTGTTCTTTTTAACACACTCGATATCG Region 3 Deficiency; CD123-Fw22:(SEQ ID NO: 151) CTTTATCCAAATAACAGTGGGAAGCCTTG CD123-Re22:(SEQ ID NO: 152) CAGTTTCTGTTGGAATGGTGGGTTGGCCACT Region 4 Deficiency;CD123-Fw23: (SEQ ID NO: 153) AGGGAGGGTACCGGTGCGGAGAATCTGACCTGCTCD123-Re23: (SEQ ID NO: 154) TCCTGAATTTGGATAGAAGAGGATCCACGTGGRegion 5 Deficiency; CD123-Fw24: (SEQ ID NO: 155)GGTCCGACGGCCCCCGCGGACGTCCAGTA CD123-Re24: (SEQ ID NO: 156)CCTCGCCCAGGTACAGCTCAAGAAATCCACGT Region 6 Deficiency; CD123-Fw25:(SEQ ID NO: 157) ACGGAACCAGCGCAGCCTTCGGTATCCCCT CD123-Re25:(SEQ ID NO: 158) TAACCAGAAAGTGGGAACTTTGAGAACC Region 7 Deficiency;CD123-Fw26: (SEQ ID NO: 159) TCTTTGATTCATTTGTCGTCTTTTCACA CD123-Re26:(SEQ ID NO: 160) ATTGGATGCCGAAGGCTGCGCTCCTGCCC

The thus obtained PCR product was subjected to 0.8% agarose gelelectrophoresis (135 V, 15 minutes, TAE buffer). DNA was visualized byethidium bromide staining. After confirming the amplification,purification was carried out using Wizard SV Gel and PCR Clean-UpSystem. The thus obtained DNA was subjected to phosphorylation usingpolynucleotide kinase (New England Biolabs) and to ethanol precipitationand then a part thereof was allowed to undergo the reaction using TaKaRaLigation Kit. Regarding the transformation, a ligation sample and DH10Bcompetent cell were mixed and spread on an LB plate (containingkanamycin). A plasmid DNA was extracted from the thus obtained colony bythe Miniprep method and digested with XhoI and NotI and the insert wasverified.

(Flow Cytometry Analysis of IL-3Rα/GM-CSFRα Chimeric Protein ExpressionCell Using Labeled Anti-IL-3Rα Antibody)

HEK293T cell was used in the preparation of IL-3Rα/GM-CSFRα chimericprotein expression cell. The plasmid DNA obtained in the above wasintroduced as an expression vector into the HEK293T. HEK293T introducedwith the expression vector was cultured under an environment of 5% CO₂and 37° C. and used in the flow cytometry analysis 2 days after theintroduction.

Each of Alexa Flour488-labeled human antibodies or commerciallyavailable FITC-labeled anti-IL-3Rα mouse antibodies (7G3 and 9F5: bothavailable from BD Biosciences, 6H6: from Acris Antibodies) at aconcentration of 1 μg/ml was allowed to react for 30 minutes on ice with100,000 to 1,000,000 cells of the chimeric protein expression cell. Astaining medium (Dulbecco's PBS containing 2% fetal calf serum, 2 mMEDTA and 0.05% NaN₃) was used for diluting the antibodies and cells.Next, the cells reacted with each antibody were washed 3 times with thestaining medium, and whether or not the labeled antibody bound to thecells was confirmed by flow cytometry.

The results are shown in FIG. 3. The reaction of the antibody c7G3disappeared only in the case of the protein expression cell in which theregion 1 was replaced by GM-CSFRα. Reaction of the Old19 disappeared inthe case of the protein expression cell in which the region 3 and region4 in A domain and region 6 and region 7 in B domain were replaced byGM-CSFRα. Reaction of the New102 disappeared in the case of the proteinexpression cell in which the region 6 and region 7 of B domain wasreplaced by GM-CSFRα.

Based on the above, it was shown a possibility that the antibody Old19recognized the regions 3 and 4 of A domain and regions 6 and 7 of Bdomain, and the antibody New102 recognized the regions 6 and 7 of Bdomain. The above results are summarized as Table 4.

TABLE 4 Region (domain) Replacing sequence 7G3 9F5 6H6 Old19 New102Region 1 55-DADYSMP-61 − ++ ++ ++ ++ (A) Region 3 91-STWILFPE-98 ++ ++++ − ++ (A) Region 4 97-PENSGKPWA- ++ ++ ++ − ++ (A-B) 104 Region 5122-CSWAVGPG- ++ ++ ++ ++ ++ (B) 128 Region 6 182-ILVRGRS-188 ++ ++ ++ −− (B) Region 7 192-GIPCTDK-198 ++ ++ ++ − − (B)

INDUSTRIAL APPLICABILITY

According to the invention, there is provided an antibody to humanIL-3Rα protein (another name: human CD123) and a therapeutic agent and adiagnostic agent for myelocytic malignant tumors, particularly acutemyeloid leukemia (AML), which comprises a human IL-3Rα antibody as anactive ingredient.

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SEQ ID NO: 3: IL-3Rα_Fw primerSEQ ID NO: 4: IL-3Rα_Re primerSEQ ID NO: 5: IL-3Rα_seqF1 primerSEQ ID NO: 6: Insert (MfeI from NotI)SEQ ID NO: 7: Rhe123Fw1 primerSEQ ID NO: 8: Rhe123Rv1 primerSEQ ID NO: 9: T7 primerSEQ ID NO: 10: SP6 primerSEQ ID NO: 11: Insert (MefI to NotI) of Macaca fascicularis IL-3RαSEQ ID NO: 12: Insert (MefI to NotI) of Macaca mulatta IL-3RαSEQ ID NO: 13: hIL-3Rαsol-FLAG-NotI primer

SEQ ID NO: 14: Insert (MefI to NotI)

SEQ ID NO: 15: hh-6 primerSEQ ID NO: 16: hh-3 primerSEQ ID NO: 17: hh-4 primerSEQ ID NO: 18: Old4 heavy chain specific primer FwSEQ ID NO: 19: Old4 heavy chain specific primer RvSEQ ID NO: 20: Old5 heavy chain specific primer FwSEQ ID NO: 21: Old5 heavy chain specific primer RvSEQ ID NO: 22: Old17 heavy chain specific primer FwSEQ ID NO: 23: Old17 heavy chain specific primer RvSEQ ID NO: 24: Old19 heavy chain specific primer FwSEQ ID NO: 25: Old19 heavy chain specific primer RvSEQ ID NO: 26: New 102 heavy chain specific primer FwSEQ ID NO: 27: New 102 heavy chain specific primer RvSEQ ID NO: 28: Old6 heavy chain specific primer FwSEQ ID NO: 29: Old6 heavy chain specific primer RvSEQ ID NO: 30: mH_Rv1 primerSEQ ID NO: 31: mH_Rv2 primerSEQ ID NO: 32: 7G3 heavy chain specific primer FwSEQ ID NO: 33: 7G3 heavy chain specific primer RvSEQ ID NO: 34: hk-2 primerSEQ ID NO: 35: hk-6 primerSEQ ID NO: 36: Old4 light chain specific primer FwSEQ ID NO: 37: Old4 light chain specific primer RvSEQ ID NO: 38: Old5 light chain specific primer FwSEQ ID NO: 39: Old5 light chain specific primer RvSEQ ID NO: 40: Old17 light chain specific primer FwSEQ ID NO: 41: Old17 light chain specific primer RvSEQ ID NO: 42: Old19 light chain specific primer FwSEQ ID NO: 43: Old19 light chain specific primer RvSEQ ID NO: 44: New 102 light chain specific primer FwSEQ ID NO: 45: New 102 light chain specific primer RvSEQ ID NO: 46: Old6 light chain specific primer FwSEQ ID NO: 47: Old6 light chain specific primer RvSEQ ID NO: 48: mK_Rv1 primerSEQ ID NO: 49: mK_Rv2 primerSEQ ID NO: 50: 7G3 light chain specific primer FwSEQ ID NO: 51: 7G3 light chain specific primer RvSEQ ID NO: 80: hCD116Fw-MfeI primerSEQ ID NO: 81: hCD116Rv-NotI primerSEQ ID NO: 82: hCD116Fw-MfeI primerSEQ ID NO: 83: hCD116Rv-NotI primerSEQ ID NO: 84: hCD116Fw-MfeI primerSEQ ID NO: 85: hCD116Rv-NotI primerSEQ ID NO: 86: hCD116SeqFw1 primerSEQ ID NO: 87: hCD116SeqFw2 primerSEQ ID NO: 88: hCD116SeqRv1 primerSEQ ID NO: 89: T7 primerSEQ ID NO: 90: hCD123-C-FLAG-R1 primerSEQ ID NO: 91: IL-3Rα_Fw primerSEQ ID NO: 92: C-FLAG-NotR2 primerSEQ ID NO: 93: pEGFP-N-1-Fw primerSEQ ID NO: 94: pEGFP-N-1-Re primerSEQ ID NO: 95: pEGFP-N-1-Fw primerSEQ ID NO: 96: pEGFP-N-1-Re primerSEQ ID NO: 97: CD123R11pEGFPN1 primerSEQ ID NO: 98: CD123F11 primerSEQ ID NO: 99: CD123R12-2 primerSEQ ID NO: 100: CD123F12-2 primerSEQ ID NO: 101: CD123R13 primerSEQ ID NO: 102: CD123F13 primerSEQ ID NO: 103: pEGFP-N-1-Fw primerSEQ ID NO: 104: pEGFP-N-1-Re primerSEQ ID NO: 105: GM-CSFRF11 primerSEQ ID NO: 106: GM-CSFRR11 primerSEQ ID NO: 107: GM-CSFRF12 primerSEQ ID NO: 108: GM-CSFRR12 primerSEQ ID NO: 109: GM-CSFRF13 primerSEQ ID NO: 110: GM-CSFRR13 primerSEQ ID NO: 111: pEGFP-N-1-Fw primerSEQ ID NO: 112: pEGFP-N-1-Re primerSEQ ID NO: 149: CD123-Fw21 primerSEQ ID NO: 150: CD123-Re21 primerSEQ ID NO: 151: CD123-Fw22 primerSEQ ID NO: 152: CD123-Re22 primerSEQ ID NO: 153: CD123-Fw23 primerSEQ ID NO: 154: CD123-Re23 primerSEQ ID NO: 155: CD123-Fw24 primerSEQ ID NO: 156: CD123-Re24 primerSEQ ID NO: 157: CD123-Fw25 primerSEQ ID NO: 158: CD123-Re25 primerSEQ ID NO: 159: CD123-Fw26 primerSEQ ID NO: 160: CD123-Re26 primer

1. An antibody to a human IL-3Rα chain, which does not inhibit IL-3signaling and binds to B domain of the human IL-3Rα chain but does notbind to C domain of the human IL-3Rα chain.
 2. The antibody according toclaim 1, further having high antibody-dependent cellular cytotoxicity(ADCC).
 3. The antibody according to claim 1, wherein the highantibody-dependent cellular cytotoxicity (ADCC) shows a specific lysisrate of 10% at an antibody concentration of 0.01 μg/ml, by aColon-26/hCD123 ADCC assay method using PBMC cultured with IL-2.
 4. Theantibody according to claim 1, which comprises amino acid sequences ofCDRs of heavy chain and CDRs of light chain selected from the groupconsisting of the following (a) to (e); (a) CDR 1 to 3 of heavy chainare the amino acid sequences of SEQ ID NOs:113 to 115, respectively, andCDR 1 to 3 of light chain are the amino acid sequences of SEQ ID NOs:131to 133, respectively, (b) CDR 1 to 3 of heavy chain are the amino acidsequences of SEQ ID NOs:116 to 118, respectively, and CDR 1 to 3 oflight chain are the amino acid sequences of SEQ ID NOs:134 to 136,respectively, (c) CDR 1 to 3 of heavy chain are the amino acid sequencesof SEQ ID NOs:119 to 121, respectively, and CDR 1 to 3 of light chainare the amino acid sequences of SEQ ID NOs:137 to 139, respectively, (d)CDR 1 to 3 of heavy chain are the amino acid sequences of SEQ ID NOs:122to 124, respectively, and CDR 1 to 3 of light chain are the amino acidsequences of SEQ ID NOs:140 to 142, respectively, and (e) CDR 1 to 3 ofheavy chain are the amino acid sequences of SEQ ID NOs:125 to 127,respectively, and CDR 1 to 3 of light chain are the amino acid sequencesof SEQ ID NOs:143 to 145, respectively.
 5. The antibody according toclaim 1, which comprises a heavy chain variable region and a light chainvariable region selected from the group consisting of the following (a)to (f); (a) a heavy chain variable region comprising an amino acidsequence from glutamine (Q) at position 20 to serine (S) at position 139in the amino acid sequence of SEQ ID NO:53 and a light chain variableregion comprising an amino acid sequence from valine (V) at position 23to lysine (K) at position 129 in the amino acid sequence of SEQ IDNO:55; (b) a heavy chain variable region comprising an amino acidsequence from glutamine (Q) at position 20 to serine (S) at position 139in the amino acid sequence of SEQ ID NO:57 and a light chain variableregion comprising an amino acid sequence from valine (V) at position 23to lysine (K) at position 129 in the amino acid sequence of SEQ IDNO:59; (c) a heavy chain variable region comprising an amino acidsequence from glutamine (Q) at position 20 to serine (S) at position 139in the amino acid sequence of SEQ ID NO:61 and a light chain variableregion comprising an amino acid sequence from aspartic acid (D) atposition 23 to lysine (K) at position 129 in the amino acid sequence ofSEQ ID NO:63; (d) a heavy chain variable region comprising an amino acidsequence from glutamine (Q) at position 20 to serine (S) at position 139in the amino acid sequence of SEQ ID NO:65 and a light chain variableregion comprising an amino acid sequence from aspartic acid (D) atposition 23 to lysine (K) at position 129 in the amino acid sequence ofSEQ ID NO:67; (e) a heavy chain variable region comprising an amino acidsequence from glutamine (Q) at position 20 to serine (S) at position 138in the amino acid sequence of SEQ ID NO:69 and a light chain variableregion comprising an amino acid sequence from aspartic acid (D) atposition 23 to lysine (K) at position 129 in the amino acid sequence ofSEQ ID NO:71 and; (f) a heavy chain variable region and/or light chainvariable region, which comprise amino acid sequences in which 1 to 3amino acid residues are deleted, substituted, added or inserted in theheavy chain variable region and/or light chain variable region shown bythe above (a) to (e).
 6. A composition for preventing or treating ablood tumor in which a cell expressing IL-3Rα is found in bone marrow orperipheral blood of a subject, which comprises the IL-3Rα antibodyaccording to claim 1 as an active ingredient.
 7. A method for treating ablood tumor in which a cell expressing IL-3Rα is found in bone marrow orperipheral blood, which comprises administering, to a subject, acomposition comprising the IL-3Rα antibody according to claim 1 as anactive ingredient.
 8. A composition for detecting a blood tumor in whicha cell expressing IL-3Rα is found in bone marrow or peripheral blood ofa biological sample from a subject, which comprises the IL-3Rα antibodyaccording to claim
 1. 9. The composition or method according to claim 1,wherein the blood tumor is acute myeloid leukemia (AML).